Background/Objectives: Frequently, healthcare-associated and community-acquired skin and soft tissue infections develop in the context of the formation of persistent, multidrug-resistant polymicrobial consortia involving both Gram-negative and Gram-positive pathogens. Non-traditional antibacterials, in particular phage- and bacteria-derived lytic enzymes, are currently the focus of attention. However, in practice their action is not sufficient for a significant effect when used as monotherapy. Here, we propose an enzyme combination approach to obtain an effective antimicrobial composition and to enhance the efficacy of enzyme-based therapy. Methods: The enzymes individual antibacterial properties and the effects of the compositions were assessed in vitro. In vivo evaluation of a combined enzymes preparation was assessed in murine wound infection against polymicrobial biofilm containing Pseudomonas aeruginosa and Staphylococcus aureus. Results: Significant synergy or additive effects of lysins combinations were shown for S. aureus strains, while indifferent or additive effect – for Gram-negative species. Treatment of mixed biofilm-colonized wounds in an animal model showed that the combination halted the chronic process and systematization of infection and promoted accelerated tissue regeneration at the wound site. Conclusions: The combinations of peptidoglycan hydrolases with different substrate specificities can significantly increase the efficacy of the composition against Gram-positive and Gram-negative bacteria resulting in improved in vitro and in vivo antibacterial properties and has a potential to treat polymicrobial infections.