A series of gemini quaternary ammonium salts (chlorides and bromides), with various hydrocarbon chain and spacer lengths, were tested. These compounds exhibited antibacterial activity against both Gram-positive and Gram-negative bacteria and were not mutagenic. The strongest antibacterial effect was observed for TMPG-10 Cl (against Pseudomonas aeruginosa ATCC 27853) and TMPG-12 Br (against Staphylococcus aureus ATCC 6538 and Escherichia coli ATCC 11229 and clinical ESBL(+) isolate 434) surfactants. These compounds inhibited the adhesion of Staphylococcus epidermidis ATCC 35984 to a polystyrene surface and eradicated biofilm formed by P. aeruginosa PAO1. The activity of studied compounds was dependent on hydrocarbon chain length.