Cronobacter sakazakii (C. sakazakii) is an opportunistic foodborne pathogen that is commonly found in infants. C. sakazakii mainly survives with the aid of biofilms, which are difficult to remove. The work described here focuses on the effect of photodynamic technology (PDT), using a 659.5 nm laser and toluidine blue O (TBO), on C. sakazakii biofilms under different incubation time, illumination time and TBO concentration. As the incubation time, TBO concentration and illumination time all increased, the sterilization rate of C. sakazakii increased first and then decreased. At an incubation time of 20 min, a TBO concentration of 50 μg/mL, and an illumination time of 30 min, the inactivation rates reached 99.92%, 99.94% and 99.99%, respectively. Response surface methodology (RSM) analysis showed that PDT had the greatest bactericidal effect on C. sakazakii at the incubation time of 20.5 min, TBO of 56 μg/mL, and the 659.5 nm laser illumination time of 30.5 min. Confocal laser scanning microscope (CLSM) and scanning electron microscopy (SEM) observation showed that PDT disrupted the biofilm structure and altered the morphology and extracellular matrix membrane of C. sakazakii, leading to their death. Results obtained from this study indicate that TBO-PDT is a promising inactivation strategy for C. sakazakii biofilm.