Acinetobacter baumannii is a nosocomial pathogen, which is a problem worldwide due to the emergence of a difficult-to-treat multidrug-resistant A. baumannii (MDRAB). Endolysins are hydrolytic enzymes produced by a bacteriophage that can be used as a potential therapeutic agent for multidrug-resistant bacterial infection in replacing antibiotics. Here, we isolated a novel bacteriophage through prophage induction using mitomycin C from clinical A. baumannii 1656-2. Morphologically, ΦAb1656-2 was identified as a Siphoviridae family bacteriophage, which can infect MDRAB. The whole genome of ΦAb1656-2 was sequenced, and it showed that it is 50.9 kb with a G + C content of 38.6% and 68 putative open reading frames (ORFs). A novel endolysin named AbEndolysin with an N-acetylmuramidase-containing catalytic domain was identified, expressed, and purified from ΦAb1656-2. Recombinant AbEndolysin showed significant antibacterial activity against MDRAB clinical strains without any outer membrane permeabilizer. These results suggest that AbEndolysin could represent a potential antimicrobial agent for treating MDRAB clinical isolates.