Endolysins are phage-encoded enzymes that cleave the peptidoglycan of host bacteria. These enzymes have gained considerable attention due to their ability to cause cell lysis, making them candidates as antibacterial agents. Most Pseudomonas aeruginosa genomes, including the common laboratory strains PAO1 and UCBPP-PA14, contain a cryptic prophage encoding a glycoside hydrolase family 19 endolysin (named PaGH19Lys in the present study). Family 19 glycoside hydrolases are known to target peptidoglycan and chitin-type substrates. PaGH19Lys was not active toward chitin but exhibited activity toward chloroform-treated Gram-negative bacteria, displaying ∼10,000-fold higher activity than hen egg white lysozyme. Analysis of products derived from PaGH19Lys activity toward purified P. aeruginosa peptidoglycan showed that the enzyme catalyzed hydrolysis of the β-1,4 linkage between N-acetylmuramic acid and N-acetyl-d-glucosamine, classifying the enzyme as a muramidase. Finally, the crystal structure of PaGH19Lys was determined and solved to 1.8 Å resolution. The structure of the enzyme showed a globular α-helical fold possessing a deep but relatively open catalytic cleft.