Bacteria in polymicrobial communities engage in complex interactions, including interbacterial antagonism. In Chromobacterium violaceum, the Type VI Secretion System (T6SS) serves as a critical antibacterial weapon, but the full set of effectors delivered by this system remains not yet completely defined. To comprehensively identify T6SS effectors, we performed co-immunoprecipitation of VgrG3, one of the six VgrGs encoded by C. violaceum, followed by LC–MS/MS analysis. Comparative proteomics revealed a diverse array of VgrG3-associated proteins, including all six VgrG homologs, phospholipase effectors from the Tle1 and Tle5 families, PAAR-domain proteins, Tap-1–like adaptors, and the Rhs-family protein RhsF. Functional analyses confirmed that RhsF acts as a T6SS-dependent antibacterial toxin neutralized by its cognate immunity protein, RhsFi (CV_1430), forming a toxin-immunity pair. X-ray crystallography of the RhsF-CT/RhsFi complex at 1.85 Å resolution revealed structural features of ADP-ribosyltransferases in RhsF and demonstrated that RhsFi inhibits toxicity by directly occluding the catalytic site. Mutagenesis assays further confirmed that RhsF toxicity depends on a catalytic triad composed of residues R1403, Y1456, and E1497. These findings expand the known repertoire of T6SS toxins in C. violaceum and place RhsF as a newly identified antibacterial effector in this bacterium.