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Aims: We investigated the fate of Salmonella in lettuce seeds grown in a hydroponic system and the potentials of applying photodynamic inactivation (PDI) to enhance microbial safety of hydroponic farming systems. Methods and Results: Lettuce was grown from Salmonella‐contaminated seeds, and rose bengal‐mediated PDI was applied. Without intervention, Salmonella could persist in plants and hydroponic farming environment throughout 6 weeks of lettuce growth. Cross‐contamination from Salmonella‐inoculated to noninoculated seedlings was observed. PDI significantly decreased Salmonella from 3.90 ± 0.31 log colony‐forming unit (CFU) per plant to 2.77 ± 0.49 log CFU per plant without extra illumination needed (p < 0.01) by week six. Conclusions: Salmonella from contaminated seeds could survive for an extended period in lettuce and hydroponic farming environment and posed serious cross‐contamination risks. Rose bengal‐mediated PDI showed promise in controlling Salmonella contamination in lettuce in a hydroponic farming setting. Significance and Impact of the Study: This study shed light on the serious food safety implications that Salmonella‐contaminated lettuce seeds might entail in a hydroponic farming environment and demonstrated rose bengal‐mediated PDI as a potential mitigation strategy. These findings contribute to the increasingly relevant field of urban farming systems and their associated food safety concerns. [ABSTRACT FROM AUTHOR]