Citrobacter spp., although frequently ignored, is emerging as an important nosocomial bacterium able to cause various superficial and systemic life-threatening infections. Considered to be hard-to-treat bacterium due to its pattern of high antibiotic resistance, it is important to develop effective measures for early and efficient therapy. In this study, the first myovirus (vB_CfrM_CfP1) lytic for Citrobacter freundii was microbiologically and genomically characterized. Its morphology, activity spectrum, burst size, and biophysical stability spectrum were determined. CfP1 specifically infects C. freundii, has broad host range (>85 %; 21 strains tested), a burst size of 45 PFU/cell, and is very stable under different temperatures (−20 to 50 °C) and pH (3 to 11) values. CfP1 demonstrated to be highly virulent against multidrug-resistant clinical isolates up to 12 antibiotics, including penicillins, cephalosporins, carbapenems, and fluroquinoles. Genomically, CfP1 has a dsDNA molecule with 180,219 bp with average GC content of 43.1 % and codes for 273 CDSs. The genome architecture is organized into function-specific gene clusters typical for tailed phages, sharing 46 to 94 % nucleotide identity to other Citrobacter phages. The lysin gene encoding a predicted D-Ala-D-Ala carboxypeptidase was also cloned and expressed in Escherichia coli and its activity evaluated in terms of pH, ionic strength, and temperature. The lysine optimum activity was reached at 20 mM HEPES, pH 7 at 37 °C, and was able to significantly reduce all C. freundii (>2 logs) as well as Citrobacter koseri (>4 logs) strains tested. Interestingly, the antimicrobial activity of this enzyme was performed without the need of pretreatment with outer membrane-destabilizing agents. These results indicate that CfP1 lysin is a good candidate to control problematic Citrobacter infections, for which current antibiotics are no longer effective.