Microorganisms such as Pseudomonas aeruginosa, Staphylococcus aureus, and Klebsiella pneumoniae are strongly associated with post-endoscopic infections due to their high capacity to form biofilms within endoscope channels. While acetic acid and sodium hypochlorite have demonstrated significant antibacterial activity against various planktonic microorganisms, data on their effectiveness against biofilms remain limited. This study aims to evaluate the biofilm-forming ability of strains isolated from endoscopes and to examine the antibiofilm effect of 3disinfectants (2%glutaraldehyde, 4% sodium hypochlorite, 4%acetic acid). Laboratory tests were performed to evaluate the efficacy of glutaraldehyde, sodium hypochlorite, acetic acid in eradicating 24-hour and 48-hour preformed biofilms of P. aeruginosa, S. aureus, and K. pneumoniae at varying contact durations (5, 10,15 minutes). S. aureus, P. aeruginosa, and K. pneumoniae are robust biofilm producers invitro. glutaraldehyde was ineffective against sessile cells. However, acetic acid eliminated 50% of the 48-hour P. aeruginosa biofilm. Sodium hypochlorite eradicate 87% of the 48-hour biofilm formed by S. aureus within 15minutes and 60% of the biofilms formed by P. aeruginosa and K. pneumoniae. These results were confirmed by scanning electron microscopy. These findings highlight the potential of sodium hypochlorite and acetic acid as viable alternatives for removal of bacterial biofilms.