This study evaluated the bioactivity and phytochemical profiles of Eutrema japonicum cultivated under two systems: land-grown wasabi leaves (LWL) and water-grown wasabi leaves (WWL), with the aim of assessing how cultivation conditions influence antibacterial and anti-quorum sensing (QS) properties. Antibacterial activity against key bacterial pathogens and QS inhibition (QSI) in Chromobacterium violaceum were examined. LWL showed significantly stronger antibacterial and QS inhibitory activities than WWL, which correlated with higher levels of all quantified compounds. The isolated compounds were further evaluated using bioactivity assays. In silico absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis indicated favorable pharmacokinetic properties and low toxicity, while molecular docking demonstrated strong interactions with bacterial proteins associated with pathogenicity. Overall, cultivation conditions appear to influence metabolite composition and bioactivity. The superior antibacterial and QSI effects of LWL extracts highlight their potential as natural anti-infective agents for food preservation and functional ingredient applications.