Continuing the search for new antimicrobial agents with antibiofilm activity, bis-quaternary ammonium compounds based on natural (3,5-dibromo-4-hydroxyphenyl)acetic acid derivatives were synthesized. Antibacterial and antibiofilm activity of ammonium salts was evaluated in vitro against S. aureus, E. coli, and P. aeruginosa, including antibiotic-resistant strains. Bis-quaternary ammonium salts 1, 5, and 6 showed antibacterial activity with MIC values ranging from 25.0 to 200.0 μg/mL. Notably, these compounds demonstrated a high level of antibiofilm activity. Ammonium salts 1 and 6 reduced biofilm formation against S. aureus ATCC 25923, P. aeruginosa PA01, E. coli ATCC 25922, and antibiotic-resistant P. aeruginosa by 92 - 97% at a concentration of 100.0 μg/mL. In addition, compounds 1 and 6 inhibited the biofilm formation of antibiotic-resistant E. coli isolate by 60% and 63%, respectively.