In this study, biofilm formation and the production of key virulence factors were systematically evaluated across 33 strains of Pseudomonas aeruginosa isolated from diverse hospital environments in Tunisia. Among these, 13 strains demonstrated strong biofilm-forming capacities. Adding glucose (9%, w/v) to the culture medium generally enhanced biofilm development, indicating that glucose supplementation may promote biofilm formation in clinical isolates. The 13 selected biofilm-forming strains exhibited a consistent production of critical virulence factors, including pyocyanin, rhamnolipid, and LasA protease, known for its staphylolytic activity. However, profiles of extracellular polysaccharide (EPS) production and motility showed considerable heterogeneity among the strains, suggesting a strain-specific regulation of these traits. Based on a comprehensive analysis of biofilm formation and virulence expression, strain 2629 was chosen as a model organism due to its robust biofilm-producing ability and high virulence factor output. This strain was used in subsequent in vitro assays to evaluate the anti-biofilm potential of a commercial disinfectant containing peracetic acid and other active agents. Results indicated that a 3% (v/v) concentration of the disinfectant, applied for 5 min, was nearly sufficient to eradicate the biofilm formed by the model strain. These findings underscore the importance of selecting a representative biofilm-forming strain for accurate in vitro assessments of disinfectant activity.