Pseudomonas aeruginosa is a formidable pathogen linked to various challenging infections. Quorum sensing (QS) plays a pivotal role in regulating the virulence factors of P. aeruginosa. Targeting QS represents a promising strategy for mitigating P. aeruginosa virulence. This study explores the potential of niclosamide (NIC) as an antivirulence agent, emphasizing its effects on biofilm formation, pigment production, and its molecular interaction with the QS regulator, LasR. A comprehensive methodology was employed, encompassing in-silico, molecular, and in vitro analyses to assess the antivirulence properties of NIC. Sub-inhibitory concentrations of NIC (64 μg/ml) were evaluated for their capacity to inhibit biofilm formation and pigment production in P. aeruginosa. NIC resulted in a 45.4 % reduction in biofilm formation, a 48.8 % decrease in pyocyanin production, and a 41.3 % reduction in rhamnolipid production. Furthermore, NIC displayed a dose-dependent antagonistic effect on LasR, with an IC50 of 5.82 ± 0.17 μM, without any noted agonistic activity. Molecular modeling and molecular dynamics (MD) simulations indicated that NIC interacts with LasR, hindering its dimerization and destabilizing its structure. These findings were corroborated by sedimentation velocity experiments and thermal shift assays. NIC shows considerable promise as an antivirulence agent against P. aeruginosa by disrupting the LasR-mediated QS system. Through its interaction with LasR, NIC inhibits biofilm formation and diminishes the production of critical virulence factors, making P. aeruginosa less virulent and more vulnerable to conventional antibiotics and immune responses.