One of the main problems for public health is the emergence of multidrug resistant (MDR) Pseudomonas aeruginosa. This work aimed to evaluate the potential anti-virulence activities of aspirin, either alone or in combination with antibiotics, against MDR P. aeruginosa isolates. In the present work, antibiotic susceptibility testing revealed high incidence of resistance among the tested P. aeruginosa isolates against aminoglycosides, antipseudomonal fluoroquinolones, and beta-lactam antibiotics. The MIC values of aspirin against tested isolates ranged from 1000 to ≥ 8000 μg/ml. The screening of our P. aeruginosa isolates for various virulence factors showed that 71% of the isolates produced biofilm. The percentage of isolates that exhibited hemolytic activity, pyocyanin production, and swarming motility was 93.5%, 71%, and 57.5%, respectively. The tested P. aeruginosa isolates produce different enzymes including protease (83%) and lipase (61%). . Also, they harbored various toxin genes, such as toxA (93%), exoT (93%), exoY (87%), exoS (67%), and exoU (32%).. Treatment with aspirin (1/4 MIC), either alone or in combination with various antibiotics, resulted in significant reductions (P-value > 0.05) in pyocyanin production, biofilm production, hemolytic activity, and swarming motility among P. aeruginosa tested isolates. Also, the treated isolates with aspirin showed significant reductions in the expression of genes: exoS (51.6%), exoY (42.7%), and exoT (33.9%). In conclusion, aspirin could be repurposed as a potential anti-virulent that interferes with P. aeruginosa pathogenicity rather than inhibiting microbial growth.