Aims:  The aim of this study was to evaluate the inactivation efficiency of Listeria monocytogenes ATCL3C 7644 and Salmonella enterica serovar Typhimurium strain DS88 by combined treatment of hypericin (Hyp)‐based photosensitization and high power pulsed light (HPPL). Methods and Results:  Cells were incubated with Hyp (1 × 10−5 or 1 × 10−7 mol l−1) in PBS and illuminated with a light λ = 585 nm. For the combined treatment, bacteria were, after photosensitization, exposed to 350 pulses of HPPL (UV light dose = 0·023 J cm−2). Fluorescence measurements were performed to evaluate optimal time for cell–Hyp interaction. Results indicate that Hyp tends to bind both Listeria and Salmonella. After photosensitization treatment, Listeria population was reduced 7 log, whereas Salmonella was inactivated just 1 log. Electron photomicrograps of Salmonella and Listeria confirmed that photosensitization induced total collapse of the Listeria cell wall, but not that of Salmonella. After combined photosensitization–HPPL treatment, the population of Listeria was diminished by 7 log and Salmonella by 6·7 log. Conclusions:  Listeria can be effectively inactivated by Hyp‐based photosensitization (7 log), whereas Salmonella is more resistant to photosensitization and can be inactivated just by 1 log in vitro. Combined treatment of photosensitization and pulsed light inactivates effectively (6·7–7 log) both the Gram‐positive and the more resistant to photosensitization Gram‐negative bacteria. Significance and Impact of the Study:  A new approach to combat Gram‐positive and Gram‐negative bacteria is proposed, combining photosensitization with high power pulsed light.

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