Background Plant-derived compounds can be used as antimicrobial agents in medicines and as food preservatives. These compounds can be applied along with other antimicrobial agents to strengthen the effect and/or reduce the required treatment dose. Results In the present study, the antibacterial, anti-biofilm and quorum sensing inhibitory activity of carvacrol alone and in combination with the antibiotic cefixime against Escherichia coli was investigated. The MIC and MBC values for carvacrol were 250 μg/mL. In the checkerboard test, carvacrol showed a synergistic interaction with cefixime against E. coli (FIC index = 0.5). Carvacrol and cefixime significantly inhibited biofilm formation at MIC/2 (125 and 62.5 μg/mL), MIC/4 (62.5 and 31.25 μg/mL) and MIC/8 (31.25 and 15.625 μg/mL) for carvacrol and cefixime, respectively. The antibacterial and anti-biofilm potential effect of carvacrol confirmed by the scanning electron microscopy. Real-time quantitative reverse transcription PCR revealed significant down-regulation of the luxS and pfs genes following treatment with a MIC/2 (125 μg/mL) concentration of carvacrol alone and of only pfs gene following treatment with MIC/2 of carvacrol in combination with MIC/2 of cefixime (p < 0.05). Conclusions Because of the significant antibacterial and anti-biofilm activity of carvacrol, the present study examines this agent as an antibacterial drug of natural origin. The results indicate that in this study the best antibacterial and anti-biofilm properties are for the combined use of cefixime and carvacrol.