Chronic infections often involve notorious pathogens like Pseudomonas aeruginosa and Staphylococcus aureus, demanding innovative antimicrobial strategies due to escalating resistance. This investigation scrutinized the antibacterial prowess of bile salts, notably taurocholic acid (TCA), ursodeoxycholic acid (UDCA), and ox bile salt (OBS), against these pathogens. Evaluations encompassed minimum inhibitory concentration (MIC) determination, scrutiny of their impact on biofilm formation, and anti-virulence mechanisms. UDCA exhibited the highest efficacy, suppressing S. aureus and P. aeruginosa biofilms by 83.5% and 78%, respectively, at peak concentration. TCA also significantly reduced biofilm development by 81% for S. aureus and 75% for P. aeruginosa. Microscopic analysis revealed substantial disruption of biofilm architecture by UDCA and TCA. Conversely, OBS demonstrated ineffectiveness against both pathogens. Mechanistic assays elucidated UDCA and TCA’s detrimental impact on the cell membrane, prompting the release of macromolecular compounds. Additionally, UDCA and TCA inhibited protease and elastase synthesis in P. aeruginosa and staphyloxanthin and lipase production in S. aureus. These results underscore the potential of UDCA and TCA in impeding biofilm formation and mitigating the pathogenicity of S. aureus and P. aeruginosa.