In this study, ten derivatives of 2-iminothiazolidin-4-one were designed and synthesized as potential antagonists of the LasR protein, guided by extensive molecular docking studies and molecular dynamics simulations (MDS). The synthesized compounds were assessed for their antimicrobial activity and the potential as quorum sensing (QS) inhibitors against Pseudomonas aeruginosa by targeting biofilm formation and associated virulence factors such as pyocyanin, rhamnolipids, protease, and hemolysin. Among these, compounds 8f, 8 g, and 8h showed significant inhibition of P. aeruginosa biofilm formation with percent inhibition of 88.87 %, 87.74 %, and 88.77 %, respectively, compared to azithromycin 66.6 %. Further studies demonstrated that 8f, 8 g, and 8h exerted QS inhibitory activity of P. aeruginosa (in sub-MIC) pyocyanin, rhamnolipid, protease, and hemolysin and rhamnolipid inhibition assays with inhibition percentages ranged from 73.86 % to 85.40 % surpassing the inhibition observed with azithromycin (54–69.91 %). Further evaluation for the most active analogs, compounds 8f, 8 g, and 8h, using an in vitro LasR inhibition assay, revealed IC₅₀ values of 1.45, 1.38 and 1.22 μM, respectively. Additionally, extensive in-silico and molecular dynamic experiments showed that compound 8f exhibited strong interactions with the LasR ligand-binding pocket, leading to the complete dissociation of the protein's dimeric form over approximately 400 ns of MDS. This confirms its action as a LasR inhibitor. These findings establish a robust functional model for LasR inhibition and highlight the potential of 2-iminothiazolidin-4-one derivatives as promising QS modulators for combating P. aeruginosa infections.