(1) Background: Limited evidence exists addressing the action of antimicrobial visible light against Cronobacter sakazakii. Here, we investigated the antimicrobial effects of blue-LED (light emitting diode) at 405 nm against two persistent dairy environment sourced strains of C. sakazakii (ES191 and AGRFS2961). (2) Methods: Beside of investigating cell survival by counts, the phenotypic characteristics of the strains were compared with a reference strain (BAA894) by evaluating the metabolic rate, cell membrane permeability, and ROS level. (3) Results: The two environment isolates (ES191 and AGRFS2961) were more metabolic active and ES191 showed dramatic permeability change of the outer membrane. Notably, we detected varied impacts of different ROS scavengers (catalase > thiourea > superoxide dismutase) during light application, suggesting that hydrogen peroxide (H2O2), the reducing target of catalase, has a key role during blue light inactivation. This finding was further strengthened, following the observation that the combined effect of external H2O2 (sublethal concentration) and 405 nm LED, achieved an additional 2–4 log CFU reduction for both stationary phase and biofilm cells. (4) Conclusions: H2O2 could be used in combination with blue light to enhance bactericidal efficacy and form the basis of a new hurdle technology for controlling C. sakazakii in dairy processing plants.
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