Two series of benzofuran-based thiosemicarbazides 7, 8(a–l) are evaluated as P. aeruginosa quorum sensing inhibitors (QSIs). The studied compounds resulted in reduction of violacein production of C. violaceum, at ¼ MIC, up to 26.04–67.36 %, relative to the reference drug azithromycin (54 %). In vitro evaluation of QS and antivirulence activities, at the same concentration, against P. aeruginosa, showed promising inhibitory effect of biofilms formation, bacterial motility and virulence factors production (pyocyanin, and rhamnolipids). Compounds 7a, 7d, and 7k inhibit biofilm formation up to 57.31 %, 67.54 %, 78.38 %, respectively, in addition to >50 % inhibition of hemolysin production. On the other hand, compounds 8a, 8d, 8f and 8k of the bromobenzofuran series showed, 55.66 %, 50.67 %, 73.34 %, 79.93 %, reduction in biofilm formation, respectively. Compounds 8d and 8f exhibit >50 % reduction of hemolysin and protease. In vivo study on mice groups infected with P. aeruginosa, previously treated with compounds 7k or 8f, revealed 50 % survival protection, accompanied by decrease in bacterial loads in kidney and liver tissues up to 50 % relative to the untreated group. Moreover, cytotoxicity study on human lung cell line (WI-38), demonstrates the safety of 7k and 8f (CC50 = 96.75 ± 3.02 and 142.4 ± 4.44, respectively). Molecular docking study into PqsRLBD, (PDB: 6b8a), showed promising interactions with the essential amino acids in the binding pockets. MD analysis revealed that compounds 8f and 7k, exhibit superior binding characteristics compared to the native inhibitor. Through a blend of structural snugness, dynamic stabilization, and energetically favorable interactions, 8f stands out as a particularly promising scaffold for the development of next-generation PqsR inhibitors.