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Conventional wound management is increasingly challenged by the rise of antimicrobial resistance and the cytotoxic side effects of traditional agents. Phototherapy, particularly using blue light (BL), offers a promising non-invasive and non-contact alternative. This study evaluated the antimicrobial efficacy and safety of a 405 nm BL-LED device against a broad spectrum of medically important wound pathogens using a collagen-based synthetic skin model, emphasizing the novelty of applying this platform for light-based antimicrobial testing. This synthetic skin provides a more realistic environment, mimicking the structure and topography of human skin. Light uniformity mapping showed consistent irradiance across a 10 cm diameter (30.14 ± 0.78 mW/cm<sup>2</sup>). The device achieved a substantial fluence-dependent microbial log reduction of up to 3.5 at 27 J/cm<sup>2</sup>, demonstrating efficacy considerably higher than that reported in similar studies. Its broad-spectrum activity was confirmed against ESKAPE pathogens and Candida albicans, a significant advantage for managing polymicrobial wound infections. A key finding was the differential susceptibility among species, with Klebsiella pneumoniae exhibiting the highest susceptibility while Staphylococcus aureus proved most resistant. This is likely due to variations in microbial envelope structure and photosensitive molecule content, which affect the reactive oxygen species (ROS). Optical transmission experiments and surface versus embedded-colony analyses revealed no statistical significance, suggesting the potential application of BL to inhibit pathogens beneath the skin. Infrared spectroscopy confirmed that BL exposure, even at high fluences (108 J/cm<sup>2</sup>), caused no detectable chemical or conformational degradation of the collagen matrix, unlike the damaging effects observed with germicidal UV-C control. Finally, a unique evaluation in an aerosol chamber demonstrated that BL treatment reduced microbial deposition on surfaces by over 95%, highlighting its potential for mitigating nosocomial infections. These findings demonstrate the suitability of skin-mimicking substrates as an accessible alternative for evaluating BL in preclinical testing, thereby eliminating the need for animal models.