The objective of this study was to evaluate the resistance of Pseudomonas fluorescens biofilms to orto-phtaldehyde action under laboratory conditions, simulating a food processing environment. Biofilms were initially formed on glass coupons. Tests were performed using different concentration of biocide, biofilms of 6 day old and a range of exposure times. The data obtained suggested that the resistance of the treated biofilms to sanitizing agent may be due to attributes of extracellular polymeric substances. The results also demonstrated that the physical stability of the biofilms has been increased with biocide application and the biocide proved to be more effective for longer exposure times.