The Use of Biomass from In Vitro Fungal Cultures as a Bioactive Ingredient with Antimicrobial Activity in Hydrogel Dressings

<b>Background/Objectives</b>: Chronic wounds represent a significant clinical burden and require multimodal treatment strategies targeting inflammation, infection, moisture balance, and tissue remodeling, as defined by the TIME framework. This study aimed to evaluate the therapeutic potential of innovative hydrogel dressings enriched with fungal biomass, designed to exploit natural bioactive compounds-such as antimicrobial peptides and proteolytic enzymes-to enhance wound healing while maintaining high biocompatibility. <b>Methods</b>: Hydrogel dressings incorporating selected fungal biomasses were fabricated and characterized for physicochemical and biological performance. Key material properties relevant to wound care, including hydrophilicity and porosity, were analyzed to assess exudate management capacity and maintenance of a moist wound environment. Antimicrobial activity was tested against common wound pathogens, and species-pathogen interactions were evaluated using generalized linear modeling. In vitro biocompatibility was assessed using human keratinocytes and compared with conventional silver nanoparticle-based dressings. <b>Results</b>: The developed hydrogels demonstrated properties suitable for clinical application, including superhydrophilicity and high porosity, supporting effective exudate control and moisture retention. Significant broad-spectrum antimicrobial activity was observed, particularly against <i>Staphylococcus aureus</i> and <i>Pseudomonas aeruginosa</i>, with effects dependent on fungal species. Statistical modeling revealed highly significant interactions between fungal species and pathogens in inhibition zones (<i>p</i> < 0.001). Hydrogels containing <i>Pleurotus ostreatus</i> and <i>Agaricus bisporus</i> showed broad activity against <i>Escherichia coli</i>, <i>P. aeruginosa</i>, and <i>S. aureus</i>, whereas <i>Enterococcus faecalis</i> exhibited resistance. Fungal biomass-based dressings displayed superior keratinocyte biocompatibility compared to silver nanoparticle controls. <b>Conclusions</b>: Fungal biomass-reinforced hydrogels offer a promising, safer, multifunctional alternative for infected chronic wound management, supporting both antimicrobial action and tissue regeneration.