*3.2. Antimicrobial Assay*

The panel of test microorganisms consisted of the following multi drug-resistant (MDR) bacteria: Gram-negative *Escherichia coli* (DSM 1116) and *Pseudomonas aeruginosa* (PA16); Gram-positive *Bacillus subtilis* (DSM 10), *Staphylococcus aureus* (DSM 346), and *Mycobacterium smegmatis* (ATCC 7000048); yeasts *Candida albicans* (DSM 1665) and *Rhodotorula glutinis* (DSM 10134); and filamentous fungi *Mucor hiemalis* (DSM 2656). The positive controls were nystatin, oxytetracyclin, kanamycin, and gentamycin, with a concentration of 1 mg/mL. Compounds **1**–**4** were prepared in a concentration of 1 mg/mL MeOH. The activity of the samples was observed by using the microdilution technique in 96 well plates for tissue cultures (TPP). As much as 20 μL of the MeOH solution of each compound (1 mg/mL) was mixed with 300 μL of bacterial or fungal suspension, except for the positive controls where 2 μL of either oxytetracycline, kanamycin, and gentamycin in 300 μL of bacterial suspension was used. The samples were tested in seven 1:2 serial dilution steps (concentrations A to H) in 96-well plates. Bacteria were cultivated in Mueller–Hinton bouillon (Roth) and fungi/yeasts in MYC medium (1.0% phytone peptone, 1.0% glucose, and 1.19% HEPES, pH 7.0). Start OD600 was 0.01 for *B. subtilis*, *E. coli,* and *S. aureus*; start OD548 was 0.1 for *M. hiemalis*, *C. albicans*, *R. glutinis*, *M. smegmatis,* and *P. aeruginosa*. The test organisms were cultivated at 30 ◦C and 160 rpm overnight.
