Interbacterial competition occurs when multiple bacteria have the ability to fulfill the same ecological niche. Vibrio fischeri is a marine bacterium that establishes a multistrain symbiosis with the Hawaiian bobtail squid, where different strains engage in competition as they colonize the light organ. One mechanism that enables this competition during symbiosis establishment is a contact-dependent molecular weapon called the type VI secretion system (T6SS). It has been well-established in other bacteria that the T6SS leads to interbacterial killing through the translocation of toxic effectors into adjacent cells. However, the specific T6SS-delivered effectors that mediate incompatibility between Vibrio fischeri strains have not been clearly identified. To address this knowledge gap, we performed cellular assays involving co-incubations of different strains to identify a set of strains that are predicted to express a functional T6SS and are also incompatible with each other. Bioinformatic analysis of the T6SS gene clusters in these strains led to the identification of a gene predicted to encode a putative effector based on the presence of conserved motifs common to lipase toxins. We called this putative effector Type VI lipase effector Vibrio fischeri 1 (TleVf1). The incompatibility observed in the cellular-based assays was consistent with our bioinformatic observations, where strains that do not encode the lipase (TleVf1-) are unable to grow well in the presence of strains that do (TleVf1+), supporting the role of TleVf1 in interbacterial competition. To further investigate the regulation of this putative effector, we constructed transcriptional reporters to compare the expression of tleVf1 with that of the T6SS structural gene hcp under environmentally relevant conditions. While hcp expression varied in response to environmental cues such as pH, tleVf1 expression remained low and unchanged across conditions, suggesting tleVf1 is not co-regulated with T6SS structural genes. This study provides insight into the molecular mechanisms that enable killing between different strains of V. fischeri, thereby increasing our understanding of factors that impact strain diversity in symbiotic populations.