The emergence of multidrug-resistant (MDR) bacteria has risen rapidly, leading to a great threat to global public health. A promising solution to this problem is the exploitation of phage endolysins. In the present study, a putative N-acetylmuramoyl-L-alanine type-2 amidase (NALAA-2, EC 3.5.1.28) fromPropionibacteriumbacteriophage PAC1 was characterized. The enzyme (PaAmi1) was cloned into a T7 expression vector and expressed inE. coliBL21 cells. Kinetics analysis using turbidity reduction assays allowed the determination of the optimal conditions for lytic activity against a range of Gram-positive and negative human pathogens. The peptidoglycan degradation activity ofPaAmi1 was confirmed using isolated peptidoglycan fromP. acnes. The antibacterial activity ofPaAmi1 was investigated using liveP. acnescells growing on agar plates. Two engineered variants ofPaAmi1 were designed by fusion to its N-terminus two short antimicrobial peptides (AMPs). One AMP was selected by searching the genomes ofPropionibacteriumbacteriophages using bioinformatics tools, whereas the other AMP sequence was selected from the antimicrobial peptide databases. Both engineered variants exhibited improved lytic activity towardsP. acnesand the enterococci speciesEnterococcus faecalisandEnterococcus faecium. The results of the present study suggest thatPaAmi1 is a new antimicrobial agent and provide proof of concept that bacteriophage genomes are a rich source of AMP sequences that can be further exploited for designing novel or improved endolysins.