The human gut microbiome and soil environments harbor dense, polymicrobial communities where frequent interbacterial interactions and contact-dependent responses occur between community members. Two phyla abundant in these niches, Gram-negative Bacteroidota and Gram-positive Bacillota, utilize contact-dependent type VI and type VII secretion systems (T6SS and T7SS), respectively, to deliver antagonistic toxins (effectors) during competitive encounters with neighboring bacteria. However, the mechanisms of interbacterial antagonism mediated by T6SS and T7SS, as well as proteomic responses to interbacterial contacts, remain underexplored in these ecosystems. Here, we reveal the widespread distribution of the T6SS- DNAse effector (Tde) and its cognate immunity protein (Tdi) amongst gut commensal Bacteroidota species and soil-derived Bacillota species. Structural and functional analyses demonstrate that Tde1 possesses nuclease activity that elicits T6SS-dependent toxicity among Bacteroidaceae strains. Despite sequence differences, the Tde proteins from Bacillota and Bacteroidota phyla exhibited high structural similarities. The crystal structures of isolated Tde and several Tde/Tdi complexes highlight a conserved inactivation mechanism across these two phyla, whereby Tdi immunity proteins bind the effector and disrupt its nuclease fold, except Enterococcus quebeciensis, which displayed slight differences with Tde C-terminus flexibility upon binding to the cognate immunity protein. The Tde/Tdi interaction mode represents a novel mechanism of inactivation distinct from other polymorphic toxin-immunity pairs of known structure. AAA family ATPases in Bacteroides ovatus are downregulated in response to contact with T6SS encoding Bacteroides strains. Deletion of these AAA ATPases alters the response to Tde delivered by T6SS, suggesting roles for these ATPases in responding to T6SS-mediated attack. Our findings reveal that horizontally transferred Tde-encoding T6SS loci confer a competitive advantage to Bacteroidales. The unique Tde/Tdi interaction suggests that Tdi homologs can provide broad immunity by disrupting the conserved Tde fold.