Objective. To determine the minimum inhibitory concentrations of thiamphenicol and thiamphenicol glycinate acetylcysteinate against clinically significant microorganisms and determine their efficacy against microbial biofilms. Materials and Methods. This study included 48 clinical strains isolated from the sputum of patients with respiratory tract infections (16 S. pneumoniae, K. pneumoniae and S. aureus strains). Antimicrobial susceptibility testing was performed using broth microdilution method. Biofilm formation culturing with antibiotics, N-acetylcysteine and their combinations was assessed in Mueller-Hinton broth and brain heart broth in 96-well plates. Biofilms are fixed with 2,5% glutaraldehyde solution, stained with 0,25% crystal violet solution, which is extracted by 33% acetic acid solution. Results. The MIC of thiamphenicol and thiamphenicol glycinate acetylcysteinate (in terms of thiamphenicol) were the same for 87,5% of strains. Thiamphenicol and thiamphenicol glycinate acetylcysteinate have been confirmed high antimicrobial activity against S. pneumoniae strains (MIC50 0,5 mg/l, MIC90 1-2 mg/l). Cultivation with chloramphenicol, thiamphenicol, thiamphenicol glycinate acetylcysteinate, combination of chloramphenicol and thiamphenicol with N-acetylcysteine contributed to significant reduction in the optical density of S. pneumoniae biofilms. Thiamphenicol increased biofilm formation in some resistant S. aureus and K. pneumoniae. Combination based on N-acetylcysteine neutralized this effect. Conclusions. Potentiation of antibacterial activity of thiamphenicol by N-acetylcysteine against S. pneumoniae biofilms has been shown.