Persistent bacterial infections are often difficult to treat as theydue to formation of biofilms, which protect microbes from host defense and reduce the efficacy of antimicrobial therapy. Trimethoprim-sulfamethoxazole (TMP-SMX), a commonly employed broad-spectrum antibiotic co-formulation, is largely effective against the majority of bacterial pathogens. Growing evidence suggests that subminimum inhibitory concentrations (sub-MICs) of antibiotics could influence the behavior of bacteria, resulting in persistence and treatment failure. This study examined the effects of subminimum doses of trimethoprim-sulfamethoxazole on biofilm formation and the possible emergence of resistance to other classes of antibiotics. Nine different bacterial strains were examined, and the results showed that all strains, except S. aureus NCTC 12393, exhibited increased biofilm formation when exposed to subminimum doses of trimethoprim-sulfamethoxazole. Biofilm formation was increased by 127.06% in Escherichia coli ATCC 14169 and decreased by 25.39% in Staphylococcus aureus NCTC 12492. Minimum inhibitory concentrations (MICs) were established by using the microbroth dilution method, and subsequently crystal violet staining was used for the examination of biofilm growth. Optical density values compared to controls were used for the categorization of biofilm-forming capacity. Additional antibiotic susceptibility tests were carried out in an attempt to seek out any possible changes in resistance profiles after exposure to TMP-SMX. These findings highlight the manner in which subinhibitory concentrations of antibiotics can significantly alter bacterial behavior, particularly in biofilm formation. This highlights a need to optimize antibiotic use and dosing, and to reevaluate TMP-SMX use in combination therapies for the treatment of chronic and biofilm-related infections.