Essential oils (EOs) have demonstrated wide-spectrum antimicrobial activities and have been actively studied for their application in foods as alternative natural preservatives. However, information regarding microbial adaptive responses and changes in virulence properties following sublethal EO exposure is still scarce. The present study investigated the effect of sublethal thymol (Thy), carvacrol (Car), or trans-cinnamaldehyde (TC) adaptation on virulence gene expression and virulence properties of Escherichia coli O157:H7. The results demonstrated that E. coli O157:H7 grown to the early stationary phase in the presence of sublethal EO showed significantly (P < 0.05) reduced motility (reversible after stress removal), biofilm-forming ability, and efflux pump activity, with no induction of antibiotic resistance and no significant changes to its adhesion and invasion ability on a human colon adenocarcinoma (Caco-2) cell line. Reverse transcription-quantitative PCR revealed reduced expression of relevant virulence genes, including those encoding flagellar biosynthesis and function, biofilm formation regulators, multidrug efflux pumps, and type III secretion system components. This study demonstrated that Thy, Car, and TC at sublethal concentrations did not potentiate virulence in adapted E. coli O157:H7, which could benefit to their application in the food industry.IMPORTANCE The present study was conducted to evaluate changes in virulence properties in Escherichia coli O157:H7 adapted to sublethal essential oils (EOs). The results demonstrated reduced motility, biofilm-forming ability, and efflux pump activities in EO-adapted E. coli O157:H7, with no induction of antibiotic resistance or infection (adhesion and invasion) on Caco-2 cells. Reverse transcription-quantitative PCR results revealed changes in the expression of related virulence genes. Thus, the present study provides new insights into microbial virulence behavior following EO adaptation and suggests that Thy, Car, and TC sublethal exposure did not constitute a significant risk in inducing microbial virulence.