Gram-negative bacteria can antagonize neighboring microbes using a type VI secretion system (T6SS) to deliver toxins that target different essential cellular features. Despite the conserved nature of these targets, T6SS potency can vary across recipient species. To understand the functional basis of intrinsic T6SS susceptibility, we screened for essentialEscherichia coli (Eco)genes that affect its survival when antagonized by a cell wall-degrading T6SS toxin fromPseudomonas aeruginosa, Tae1. We revealed genes associated with both the cell wall and a separate layer of the cell envelope, lipopolysaccharide, that modulate Tae1 toxicityin vivo. Disruption of genes in early lipopolysaccharide biosynthesis providedEcowith novel resistance to Tae1, despite significant cell wall degradation. These data suggest that Tae1 toxicity is determined not only by direct substrate damage, but also by indirect cell envelope homeostasis activities. We also found that Tae1-resistantEcoexhibited reduced cell wall synthesis and overall slowed growth, suggesting that reactive cell envelope maintenance pathways could promote, not prevent, self-lysis. Together, our study reveals the complex functional underpinnings of susceptibility to Tae1 and T6SS which regulate the impact of toxin-substrate interactionsin vivo.