Aims: The objective of this study was to determine the combined effect of thymoquinone (TQ) and mild heat on Cronobacter sakazakii in reconstituted infant formula. Methods and Results: Reconstituted infant formula samples inoculated with a mixture of four C. sakazakii strains (approx. 6.5 log CFU ml(-1)) were prepared with various concentrations of TQ (0, 5, 10, 20 and 30 mmol l(-1)) and were heated to 45, 50 and 55 degrees C for 0, 10, 20, 30, 60 and 120 min, and the surviving populations of C. sakazakii at each sampling time were enumerated. To elucidate the mode of action of TQ, membrane integrity and changes in cell morphology were examined by LIVE/DEAD((R)) BacLight (TM) bacterial viability kit and field emission scanning electron microscope respectively. TQ at 30 mmol l(-1) reduced the pathogen to undetectable level in between 60 and 120 min at 45 degrees C, 60 min at 50 degrees C and 10 min at 55 degrees C respectively. Conclusions: Our results demonstrated that the combined treatments significantly reduced (P < 0.05) the population of C. sakazakii, compared to the control. Cronobacter sakazakii numbers were reduced much more rapidly with higher temperatures and increased concentrations of TQ. And combined treatment inactivated pathogen partly by causing cell membrane disruption. Significance and Impact of the Study: These findings suggested that TQ, together with mild heat, may have potential application in infant formula to control C. sakazakii before consumption and therefore is a possible way to prevent infections associated with C. sakazakii in infant formula.