*2.5. Fungicidal Microdilution Broth Assay (96-Microplate)*

This assay was carried out to determine the minimum fungicidal concentration (MFC) which is defined as the lowest concentration of the tested antimicrobial agent that can inhibit the growth of fungi significantly differently to the growth of the negative control, as reported by Arikan [23]. The results of the fungicidal effect of mint EO and its main single constituents on mycelium growth percentage are reported in Table 3, whereas the MFC values of peppermint EO and its two main constituents are reported in Table 4 using the tendency-line formula of the chart in Microsoft Excel. The studied EO showed 4.78, 2.91, 5.40 and 4.98 mg/mL, corresponding to the inhibition of 50% visible growth of fungal mycelium of *M. fructicola*, *B. cinerea*, *A. niger* and *P. expansum*, respectively.

Regarding menthol, the MFC values were 0.85, 1.40, 1.45 and 1.21 mg/mL against *M. fructicola*, *B. cinerea*, *A. niger* and *P. expansum*, respectively. In the case of menthone, the MFC values were 1.31, 1.37, 1.90 and 1.69 mg/mL, against *M. fructicola*, *B. cinerea*, *A. niger* and *P. expansum*, respectively.


**Table 3.** Fungicidal effect of EO and single constituents on mycelium growth (%) in broth culture.

Values followed by different letters in each column for each tested fungi are significantly different at *p* < 0.05 according to one-way ANOVA combined with *Tukey* B post hoc test. (\*) are the mycelium growth percentages corresponding to the MFC values. Data are expressed as the mean of three replicates ± SD and presented for peppermint EO and the two single substances.

**Table 4.** MFC values of fungicidal effect of studied EO and single constituents.

