A new, optimized, antimicrobial enzyme system was developed for the control of wound bioburden. This Glucose oxidase-Lactoperoxidase-Guaiacol (GLG) system was analyzed for antimicrobial activity and cytotoxicity. The susceptibility of a wide range of antibiotic-resistant bacterial strains to the GLG-enzyme system was analyzed using minimum inhibitory concentration (MIC90), minimum bactericidal concentration (MBC) determination, and growth kinetics analysis. Additionally, challenge tests and cytotoxicity tests were performed with a new hydroactive alginate gel dressing with antimicrobial activity obtained by the presence of the GLG-enzyme system (Flaminal® Forte, Flen Pharma, Kontich, Belgium). All bacterial strains were susceptible to the GLG-enzyme system at low concentrations. The exact concentration required for growth arrest and cell death was dependent on the experimental design. Further, a 20% (w/v) GLG dilution showed no cytotoxicity toward fibroblasts and keratinocytes. Conversely, other antimicrobial wound-care products applied with the same dilution showed a high degree of cytotoxicity. With increasing concerns about bacterial resistance to antibiotics, this study shows that low concentrations of the GLG-enzyme system are successful in killing antibiotic-resistant bacterial strains. Furthermore, results show that GLG-enzyme system combines strong antimicrobial activity with non-cytotoxicity and promotes optimal wound healing.