The bacterial resistance is an increasing obstacle against the global health that necessitates innova‑ tion of new approaches. Targeting bacterial resistance is one of the promising approaches. Pseudomonas aeruginosa is a clinically significant opportunistic pathogen and causes wide diverse of illness. The P. aeruginosa virulence is regu‑ lated by several systems as quorum sensing (QS) systems. Additionally, P. aeruginosa could employ membranal sensors to sense the neurotransmitters enhancing the virulence. Fluoxetine (FLU), an antidepressant, functions by inhibiting the reuptake of the neurotransmitter serotonin. This study aimed to assess the anti-virulence activity of FLU against P. aeruginosa. The effect of FLU at sub-inhibitory concentration was evaluated on the biofilm formation, removal of pre‑ formed biofilms, production of virulence factors such as protease, hemolysins, elastase, rhamnolipids, motility, pyocya‑ nin, and pyoverdine. The impact of FLU on the expression of virulence-related genes was estimated. An invasion assay and mice protection assay were conducted to assess the FLU’s diminishing effect on P. aeruginosa pathogene