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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., (© 2021 The Society for Applied Microbiology.)