The type VI secretion system (T6SS) is a contact-dependent bacterial weapon to inject hazardous proteins into competitors and can be pivotal for bacterial fitness. Throughout the lifecycle of Vibrio cholerae, beneficial and detrimental activities of the T6SS have been described, but its role in biofilm-associated interactions remains poorly understood. Here, we show that biofilm formation is accompanied by significant repression of T6SS genes. Expression analyses across multiple V. cholerae isolates revealed consistent downregulation of the major T6SS gene cluster in biofilm-derived cells relative to planktonic cultures. Consistent with this biofilm-dependent repression, a loss of the T6SS did not affect V. cholerae biofilm formation. Notably, biofilm-derived V. cholerae wild-type isolates and their isogenic T6SS-deletion mutants displayed comparable resistance to attacks by T6SS-reactive Pseudomonas aeruginosa. These findings suggest that T6SS repression during biofilm growth provides a fitness advantage for V. cholerae by limiting susceptibility to T6SS-mediated counterattacks from neighboring heterologous T6SS⁺ predatory species. Our results, therefore, identify conditional T6SS silencing as a potential adaptive strategy that promotes survival within multispecies microbial communities, particularly in environments containing bacteria with highly active or more potent T6SS machineries.