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. 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 6weeks of lettuce growth. Cross-contamination from Salmonella-inoculated to noninoculated seedlings was observed. PDI significantly decreased Salmonella from 3.90±0.31log colony-forming unit (CFU) per plant to 2.77±0.49log CFU per plant without extra illumination needed (p<0.01) by week six. 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. 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.