New analogues of pulmonarin B were synthesized from (3,5-dibromo-4-hydroxyphenyl)acetic acid derivatives, and their antibacterial and antibiofilm activities against E. coli, S. aureus, and P. aeruginosa were evaluated. The antibacterial activity of synthesized ammonium salts against the methicillin-resistant strain of S. aureus 222 depends on the length of the alkyl chain. Bisquaternary ammonium salt 5 demonstrated better activity against S. aureus 222, E. coli 311, and P. aeruginosa 449 compared to mono-alkylated derivatives. Analogues of pulmonarin B 5 and 4d reduced S. aureus 222 biofilm formation by 74.5% and 89.4%, respectively. In addition,
compound 4c turned out to be the most active against the biofilm formation of P. aeruginosa 449 (biomass decreased by 39.8%).