*L. monocytogenes* and *E. coli*

The concentration used for cinnamon was 0.1–2 mg/mL and for trans-cinnamaldehyde 0.06–0.5 mg/mL. After biofilm formation for 24 h, the cell number of control samples was 11 log CFU/mL for *L. monocytogenes* and 5 log CFU/mL for *E. coli*. Treatment with cinnamon at concentrations higher than 0.1 mg/mL decreased the biofilm formation (CFU: 10 log CFU/mL for both bacteria) (Figure 1A). At 0.25 mg/mL (MIC of *E. coli*), both bacteria grew up to 10 log CFU/mL each. At 0.5 mg/mL concentration (double the MIC for *E. coli* and half the MIC for *L. monocytogenes*, see Table 1), a monoculture biofilm appeared with *Listeria* being absent. From a concentration of 1 mg/mL (MIC for *L. monocytogenes*, see Table 1), no survivors of either bacterium were detected. Trans-cinnamaldehyde exhibited similar activity against biofilm formation as compared to cinnamon (Figure 1B).

After treatment with marjoram (concentration range: 0.5–8 mg/mL) and the component terpinen-4-ol (concentration range: 1–8 mg/mL), cell number analysis showed that *E. coli* was eliminated from the biofilm at the lowest concentration, so that this culture contained *L. monocytogenes* cells only (*p* < 0.001). Biofilm elimination started form the lowest concentrations used (0.5 mg/mL for marjoram and 1 mg/mL for terpinene-4-ol) (Figure 1C,D).

Finally, thyme EO (concentration range: 0.5–4 mg/mL) exhibited strong inhibitory effect against the *L. monocytogenes* and *E. coli* mixed culture biofilm, at all the concentrations investigated (*p* = 0.03) (Figure 1E). Moreover, no survivors were detected at 1 mg/mL (half the MIC for *L. monocytogenes*, see Table 1). In addition, thymol (concentration range: 0.1–1 mg/mL) inhibited biofilm formation from its lowest concentration (Figure 1F). Surviving cells were undetectable at the sub-MIC value of 0.2 mg/mL.

**Figure 1.** Effect of essential oils (EOs) on the biofilm formation of *Listeria monocytogenes* and *Escherichia coli* polymicrobial cultures. (**A**) cinnamon EO, (**B**) cinnamaldehyde, (**C**) marjoram EO, (**D**) terpinen-4-ol, (**E**) thyme EO, and (**F**) thymol. Columns represent the OD590 values, dashed lines represent cell numbers of *L. monocytogenes*, and dotted lines cell numbers of *E. coli*. Results are presented as mean ± standard deviation of six replicates. Different letters indicate statistically significant differences between columns (*p* < 0.05).

#### *L. monocytogenes* and *S. aureus*

In the control sample, 7 log CFU/mL was recorded for both bacteria after biofilm formation for 24 h. All EOs and their components significantly decreased biofilm formation (Figure 2). The results indicate that only concentrations higher than the MIC values were effective at eliminating the bacteria. At 8 mg/mL (double the MIC for *Listeria* and more than double the MIC for *S. aureus*, see Table 1), marjoram EO (concentration range: 1.6–8 mg/mL) eliminated *Listeria* from the mixed culture, but *S. aureus* was still present (Figure 2E). At the same concentration, terpinen-4-ol (concentration range: 1.6–8 mg/mL) reduced the CFU of both bacteria to an undetectable level (Figure 2F). Similar results were obtained with cinnamon (concentration range: 0.2–2 mg/mL) and its component trans-cinnamaldehyde (concentration range: 0.1–0.8 mg/mL) (Figure 2A,B). Moreover, thyme (concentration range: 0.4–4 mg/mL) reduced polymicrobial biofilms from a concentration of 0.4 mg/mL (MIC for *L. monocytogenes*) (Figure 2C). Finally, thymol (concentration range: 0.2–1.5 mg/mL) reduced the biofilm investigated and killed both bacteria from a concentration of 0.2 mg/mL (MIC for *Listeria*) (Figure 2D).

**Figure 2.** Effect of EOs on the biofilm formation of *Listeria monocytogenes* and *Staphylococcus aureus* polymicrobial cultures. (**A**) cinnamon EO, (**B**) trans-cinnamaldehyde, (**C**) thyme EO, (**D**) thymol, (**E**) marjoram EO, and (**F**) terpinen-4-ol. Columns represent the OD590 values, dashed lines represent cell numbers of *L. monocytogenes*, and dotted lines cell numbers of *S. aureus*. Results are presented as mean ± standard deviation of six replicates. Different letters indicate statistically significant differences between columns (*p* < 0.05).

#### *L. monocytogenes* and *P. putida*

Figure 3 shows that the number of *Listeria* cells was 8 log CFU/mL and that of *P. putida* was 7 log CFU/mL in the control samples. Concerning cinnamon EO (concentration range: 0.1–1 mg/mL) and trans-cinnamaldehyde (concentration range: 0.1–1 mg/mL), significant reduction in biofilm formation was observed at 0.1 mg/mL; although higher concentrations up to 0.5 mg/mL did not achieve better inhibition (Figure 3A, B). Thyme EO (concentration range 1–20 mg/mL) inhibited the formation of *L. monocytogenes* and *P. putida* co-cultured biofilm as well, at 1 mg/mL (Figure 3C). However, *P. putida* was present (4 log CFU/mL) in the biofilm even at the highest concentration of thyme (20 mg/mL) (Figure 3C), which was equal to the MIC against this bacterium (Table 1). Thymol was applied between 0.1–1 mg/L where biofilm inhibition and decrease in CFU started at the lowest concentration used.

**Figure 3.** Effect of EOs and EO main components on the biofilm formation of *Listeria monocytogenes* and *Pseudomonas putida* polymicrobial cultures. (**A**) cinnamon EO, (**B**) trans-cinnamaldehyde, (**C**) thyme EO, (**D**) thymol, (**E**) marjoram EO, and (**F**) terpinen-4-ol. Columns represent the OD590 values, dashed lines represent cell numbers of *L. monocytogenes*, and dotted lines cell numbers of *P. putida*. Results are presented as mean ± standard deviation of six replicates. Different letters indicate statistically significant differences between columns (*p* < 0.05).

Marjoram EO (concentration range: 1–8 mg/mL) decreased the number of *P. putida* cells at 8 mg/mL by only 2 log CFU/mL compared with the control (double the MIC for *Listeria* and four times the MIC for *P. putida*), (Figure 3E). Meanwhile terpinene-4-ol (concentration range: 1–8 mg/mL) eliminated both bacteria from the biofilm at 8 mg/mL and had similar biofilm inhibitory effect to the parent oil.
