Background and Purpose: Biofilms on the surfaces of urinary catheters are among the pivotal factors for recurrent and persistent infections in urology. Many techniques have been investigated and applied for eradication of these biofilms-but with no full success. The aim of this study was to examine the effect of sustained release medicated varnish, releasing chlorhexidine, on the formation of biofilm on the urinary catheter surface in an in-vitro model. Materials and Methods: A batch model was used to test the antibacterial/ antibiofilm effect of the sustained release varnish: Catheter pieces coated with sustained release varnishes were placed in bacterial growth medium that was infected with Pseudomonas aeruginosa for 96 hours. Various concentrations of chlorhexidine impregnated in the varnish were tested. After the incubation period, the catheter pieces were assessed for biofilm formation by measuring the optical density, colony-forming units, and using confocal laser scanning microscopy, and electron scanning microscopy. Results: Biofilm growth measurement (colony-forming units [CFU]) on the catheter surface coated with the various concentrations of chlorhexidine in sustained released varnish revealed a 94% reduction with 1% chlorhexidine (P<0.0001) and 43% reduction with 0.1% chlorhexidine (P=0.08) coated varnish in comparison with a positive control or the placebo varnish in preventing biofilm growth of P. aeruginosa. These biologic assays were confirmed using confocal and electron microscopy. Conclusions: Of the various tested concentrations of sustained release varnishes, the 1% chlorhexidine concentration has demonstrated the superior antibiofilm effect on urinary catheters with P. aeruginosa. Although similar varnishes are used in dentistry, it needs extended research in animals before applying this technology in human trials.