Objective To analyze the effect of two instruments on the surface roughness (Ra) and bacterial adhesion of endoscope forceps channel. Methods Equal length sections of new polytetrafluoroethylene (Teflon) tubes were bent and fixed. Cleaning brush (Group A) and biopsy forceps (Group B) were used to simulate clinical operations with different passing frequencies. Surface roughness (Ra) and morphology were measured. Pseudomonas aeruginosa was inoculated onto the Teflon tubes in each group to evaluate surface colony-forming units and bacterial adhesion. Results Teflon tube surfaces without instrument passage were smooth and without scratches. As the passage frequency increased, surface scratches became denser, with more significant effects observed in Group A. At passage frequencies of 0, 50, 100, and 200, there were no statistically significant differences in Ra between Groups A and B. However, at a passage frequency of 500, Ra in Group A was greater than in Group B (P < 0.01). When comparing different passage frequencies within each group, there were no statistically significant differences between 0 and 50 passages, but Ra at 0 passages was significantly lower than at 100, 200, and 500 passages (P < 0.01). Initially, biofilms were evenly distributed on the Teflon tube surfaces without instrument passage, but with increased passage frequency, Group A showed more densely covered biofilms, with visible bacterial colonies at the scratch sites. There were no significant differences in colony counts between Groups A and B at 0, 50, 100, and 200 passage frequencies. However, at 500 passages, Group A had a higher colony count than Group B (P < 0.01). When comparing the colony counts at 50, 100, and 200 passage frequencies with those at 0 passages within each group, no statistically significant differences were found (P> 0.05). However, at 500 passages, the mean colony count in Group A was higher than at 0 passages (P < 0.01), while no statistically significant difference was observed in Group B compared to 0 passages (P=0.186). Conclusion Repeated passage of endoscope cleaning brush and biopsy forceps through forceps' channels increases the Ra of the endoscope forceps' channels, leading to an increase in bacterial adhesion. The effect is more significant with the cleaning brush. This highlights the need to strengthen the management of instrument usage in clinical settings and develop better cleaning tools.