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Introduction Acetylsalicylic acid (ASA) is one of the promising candidates for enhancing the action of antibacterial drugs. Purpose To evaluate in vitro the effect of aspirin in combination with antibacterial drugs on the growth of microbial cultures of clinically significant bacterial strains in osteomyelitis. Materials and methods The object of the study is clinically significant bacterial strains K. pneumoniae ( n = 12), P. aeruginosa ( n = 12), S. aureus ( n = 12), S. epidermidis ( n = 12), isolated from wounds of patients with chronic osteomyelitis. The experiment was divided into four series: control group; group of ASA administration; group of antibiotic administration; group of ASA + antibiotic administration. Antibacterial effect was assessed by the absence of a bacterial growth zone around the discs. Results There was no growth of microorganisms on Petri dishes when ASA (0.05 %) was incubated with gram-negative bacteria (K. pneumoniae , P. aeruginosa ). In the case of S. aureus bacteria, the number of CFU was reduced compared to the control group. There was no growth on plates with S. epidermidis. When polymyxin was incubated with P. aeruginosa , slight bacterial growth was recorded on Petri dishes, while only individual colonies were observed with K. pneumoniae. When incubated together with polymyxin and aspirin (0.03 %), there was no growth of microorganisms on plates with K. pneumoniae, while slight growth was noted on plates with P. aeruginosa . Gentamicin suppressed the growth of S. aureus bacteria (an insignificant number of bacteria was noted on the plates). ASA (0.03 %) enhanced the bactericidal properties of gentamicin (there was no bacterial growth on the plates). Discussion The absence of growth on plates with S. epidermidis bacteria under the influence of ASA, even without antibiotics, suggests that one of the mechanisms of its action may be the suppression of adhesion and biofilm formation. The presence of a pronounced effect of ASA on gram-negative bacteria (K. pneumoniae , P. aeruginosa ) may be associated with the effect on the outer membrane of microbial cells, while aspirin had a lesser effect on gram-positive bacteria (S. aureus ) and only reduced metabolic activity. Conclusion Our study demonstrated that acetylsalicylic acid exhibits synergism with a number of antibiotics in vitro and significantly enhances their bactericidal activity against clinical isolates associated with osteomyelitis.