Aims To study the mechanism of bacterial inactivation by carvacrol and the influence of genetic and environmental factors in its antimicrobial activity. Methods and Results In general, bacterial inactivation by carvacrol was higher in the Gram-positive Listeria monocytogenes than in the Gram-negative Escherichia coli and at acidic pH. At pH 4.0, 25 mu l l-1 of carvacrol for 5 h inactivated 1 and more than 5 log10 cycles of E. coli and L. monocytogenes populations, respectively. Genetic and environmental factors also influenced cell resistance to carvacrol: rpoS and sigB deletion decreased carvacrol resistance in E. coli and L. monocytogenes, respectively; a heat shock induced a phenomenon of cross-protection to carvacrol treatments. Repair of sublethal injuries in cell envelopes suggested that carvacrol targets lipid fractions and proteins of these structures. This result was corroborated by attenuated total reflectance infrared microspectroscopy analysis. Conclusions This study shows critical genetic and environmental factors, such as rpoS or sigB and heat shocks, and reveals new microbial structures involved in the mechanism of bacterial inactivation by carvacrol. Significance and Impact of the Study A better understanding of the mechanisms of microbial inactivation is of great relevance to design more appropriate carvacrol treatments with high antimicrobial effects.