Cell autolysis could lead to a decrease in both cell viability and the production of biochemicals, presenting one of the significant challenges during fermentation. Bacillus licheniformis, a gram-positive bacterium widely used in the production of various biologic products, also confronts the limitation caused by cell autolysis. In this study, we investigated the impact of peptidoglycan hydrolases (LytC, LytD, LytE, CwlC), endopeptidase inhibitor IseA, and prophage gene xpf on cell growth and biochemical synthesis in B. licheniformis DW2. The results showed that the deletion of xpf and overexpression of iseA could significantly increase cell survival. Then, xpf was deleted on iseA overexpressed strain P