Aroylhydrazones 1–13 were screened for antimicrobial and antibiofilm activities in vitro. N′-(2-hydroxy-phenylmethylidene)-3-pyridinecarbohydrazide (2), N′-(5-chloro-2-hydroxyphenyl-methylidene)-3-pyridinecarbohydrazide (10), N′-(3,5-chloro-2-hydroxyphenylmethylidene)-3-pyridinecarbohydrazide (11), and N′-(2-hydroxy-5-nitrophenylmethylidene)-3-pyridinecarbohydrazide (12) showed antibacterial activity against Escherichia coli, with MIC values (in µmol mL−1) of 0.18–0.23, 0.11–0.20, 0.16–0.17 and 0.35–0.37, resp. Compounds 11 and 12, as well as N′-(2-hydroxy-3-methoxyphenylmethylidene)-3-pyridinecarbohydrazide (6) and N′-(2-hydroxy-5- methoxyphenylmethylidene)-3-pyridinecarbohydrazide (8) showed antibacterial activity against Staphylococcus aureus, with the lowest MIC values of 0.005–0.2, 0.05–0.12, 0.06–0.48 and 0.17–0.99 µmol mL−1. N′-(2-hydroxy-5-methoxyphenylmethylidene)-3-pyridinecarbohydrazide (7) showed antifungal activity against both fluconazole resistant and susceptible C. albicans strains with IC
90 range of 0.18–0.1 µmol mL−1. Only compound 11 showed activity against C. albicans ATCC 10231 comparable to the activity of nystatin (the lowest MIC 4.0 ×10−2
vs. 1.7 × 10−2 µmol mL−1). Good activity regarding multi-resistant clinical strains was observed for compound 12 against MRSA strain (MIC 0.02 µmol mL−1) and compounds 2, 6 and 12 against ESBL+ E. coli MFBF 12794, with the lowest MIC for compound 12 (IC
50 0.16 µmol mL−1). Anti-biofilm activity was found for compounds 2 (MBFIC 0.015–0.02 µmol mL−1 against MRSA) and 12 (MBFIC 0.013 µmol mL−1 against EBSL+ E. coli). In the case of compound 2 against MRSA biofilm formation, MBFIC values were comparable to those of gentamicin sulphate, whereas in the case of compound 12 and EBSL+ E. coli even more favourable activity compared to gentamicin was observed.