Salmonella enterica is a Gram-negative orofecal transmitted pathogen that causes a wide diversity of local and systemic illnesses. Salmonella enterica utilizes several interplayed systems to regulate its invasion and pathogenesis: namely, quorum sensing (QS) and type three secretion system (T3SS). In addition, S. enterica could sense the adrenergic hormones in the surroundings that enhance its virulence. The current study aimed to evaluate the ability of α-adrenoreceptor antagonist prazosin to mitigate the virulence of S. enterica serovar Typhimurium. The prazosin effect on biofilm formation and the expression of sdiA, qseC, qseE, and T3SS-type II encoding genes was evaluated. Furthermore, the prazosin intracellular replication inside macrophage and anti-virulence activity was evaluated in vivo against S. typhimurium. The current finding showed a marked prazosin ability to compete on SdiA and QseC and downregulate their encoding genes. Prazosin significantly downregulated the virulence factors encoding genes and diminished the biofilm formation, intracellular replication inside macrophages, and in vivo protected mice. To sum up, prazosin showed significant inhibitory activities against QS, T3SS, and bacterial espionage, which documents its considered anti-virulence activities.