The overuse and misuse of antibiotics in treating bacterial infections cause the rapid emergence of drug-resistant bacteria, suggesting that the development of alternative strategies to control antibiotic-resistant bacteria is urgently needed. Endolysins are bacteriophage-encoded enzymes that can degrade peptidoglycan in bacterial cell walls, and they have great potential as alternative antimicrobial agents. However, exogenous application of recombinant endolysin is limited to Gram-positive bacteria because endolysins cannot penetrate the outer membrane of Gram-negative bacteria. Here, a liposome-mediated endolysin encapsulation system was developed, and its ability to penetrate the outer membrane of Gram-negative bacteria was tested. The phage-derived endolysin BSP16Lys was isolated, characterized, and used for encapsulation into a cationic liposome comprised of dipalmitoylphosphatidylcholine (DPPC), cholesterol, and hexadecylamine. The BSP16Lys-encapsulated liposome had a high zeta potential value (over 30 mV) with an average diameter of 303 nm. The encapsulation efficiency of BSP16Lys into the liposome was 35.27%. Salmonella Typhimuriumand Escherichia coli cells treated with BSP16Lys-encapsulated liposomes showed 2.2-log CFU/mL and 1.6-log CFU/mL reductions in the viable cell numbers, respectively, without treatment of a membrane permeabilizer. These results showed potential for liposome-mediated delivery of endolysin for exogenous application against Gram-negative bacteria.