Benzalkonium chloride (BAC) is a chlorine-based chemical compound with proven antimicrobial properties against bacteria, viruses, and fungi, depending on the length of the alkyl chain. It can be used as a biocide, as a cationic surfactant, and as a phase transfer agent. The aim of this study was to investigate the effectiveness of commercial cleaning agents for sanitary areas Bis duo Active (BDA) with BAC as an active compound in working concentrations of 5% and 20% on the destruction of bacterial biofilm formed on ceramic tiles. A biofilm of Staphylococcus aureus, Salmonella enterica serotype Typhimurium, and Listeria monocytogenes were grown on ceramic tiles with dimensions of 2.5 × 2.5 cm over 24 h. These plates were then treated with working concentrations of BAC for 10 min. After washing, ATP bioluminescence was measured with a luminometer, CFU/cm2 was determined and the total biomass reduction was measured after crystal-violet staining. Both working concentrations of benzalkonium chloride proved to be very effective in destroying the created bacterial biofilm on ceramic tiles. Both treatments caused a reduction in total bacteria number of up to 3.12 log10CFU/cm2, followed by a reduction in total biomass up to circa (ca.) 64%. Inhibition rates varied from ca. 28% to ca. 99%, depending on the method and concentration used. Mechanical cleaning prior or after treatment with BAC is essential to improve biofilm destruction. All methods used in this study are effective for the determination of the anti-biofilm activity of BAC. Further analyses are needed.