This study aimed to compare the antimicrobial, anti-biofilm, and cytotoxic effects of thymol/ampicillin with thymol/cefotaxime on Escherichia coli (E. coli). The antimicrobial investigation of thymol / cefotaxime and thymol/ampicillin individually and in combination was performed on E.coli ATCC25922 bacterial strain and 20 clinical strains by MIC-MBC method. Biofilm skill, kinetic kinetics, checkerboard method of these compounds were performed on E.coli ATCC25922 bacterial strain. The investigation of the functional groups and chemical bands of these compounds was done with FTIR device, and the toxicity investigation was done on human red blood cells by hemolysis method and skin fibroblast cells by MTT method. The results of this study showed that the antimicrobial effects of the thymol/ampicillin 2/8) µg/ml) were better than the thymol/cefotaxime 16/128) µg/ml( in both ATCC and clinical strains. In the examination with the FTIR device, one band named C=C conjugated, C≡C in both compounds showed the connection between thymol/ampicillin and thymol/cefotaxime. The biofilm inhibition effect of thymol/ampicillin (62 %) was better than thymol/cefotaxime (39.28 %) on E. coli ATCC25922. The bacterial killing time curve of the thymol/ampicillin at a lower concentration and time was better than the combination of thymol/cefotaxime. Cytotoxicity of synergistic compounds on RBCs and human skin fibroblasts (Ffk) was not different and was lower than that of Triton X-100. This study showed that the antimicrobial and anti-biofilm effects of the combination of thymol/ampicillin are better than the combination of thymol/cefotaxime, which can be used as a new drug in the treatment of diseases caused by E. coli bacteria.