ABSTRACT Beta-hemolytic Streptococcus agalactiae is the leading cause of bacteremia and invasive infections. These diseases are treated with β-lactams or macrolides, but the emergence of less susceptible and even fully resistant strains is a cause for concern. New bacteriophage lysins could be promising alternatives against such organisms. They hydrolyze the bacterial peptidoglycan at the end of the phage cycle, in order to release the phage progeny. By using a bioinformatic approach to screen several beta-hemolytic streptococci, a gene coding for a lysin was identified on a prophage carried by Streptococcus dysgalactiae subsp. equisimilis SK1249. The gene product, named PlySK1249, harbored an original three-domain structure with a central cell wall-binding domain surrounded by an N-terminal amidase and a C-terminal CHAP domain. Purified PlySK1249 was highly lytic and bactericidal for S. dysgalactiae (2-log ₁₀ CFU/ml decrease within 15 min). Moreover, it also efficiently killed S. agalactiae (1.5-log ₁₀ CFU/ml decrease within 15 min) but not several streptococcal commensal species. We further investigated the activity of PlySK1249 in a mouse model of S. agalactiae bacteremia. Eighty percent of the animals ( n = 10) challenged intraperitoneally with 10 ⁶ CFU of S. agalactiae died within 72 h, whereas repeated injections of PlySK1249 (45 mg/kg 3 times within 24 h) significantly protected the mice ( P < 0.01). Thus, PlySK1249, which was isolated from S. dysgalactiae , demonstrated high cross-lytic activity against S. agalactiae both in vitro and in vivo . These encouraging results indicated that PlySK1249 might represent a good candidate to be developed as a new enzybiotic for the treatment of systemic S. agalactiae infections.