A probiotic-based microneedle (MN) patch with a 5% glycerol (GL) matrix was developed to accelerate the healing of infected wounds. The MN patch contains Lactobacillus reuteri (L. reuteri), which can metabolize glycerol to produce reuterin, a broad-spectrum antibacterial substance. The MN patch provides a long-acting antibacterial and anti-inflammatory effect, with L. reuteri remaining viable for over 60 days at 4°C. In a mouse model of Staphylococcus aureus-infected wounds, the 5% GL MN patch showed superior antimicrobial efficiency and wound healing performance compared to control groups, indicating its potential for treating chronic infections. The MN patch was fabricated using a vacuum-assisted micromolding method, with different glycerol concentrations tested for optimal performance. The 5% GL MN patch demonstrated efficient delivery of L. reuteri, rapid dissolution after skin insertion, and effective inhibition of bacterial growth. In vitro studies showed that the MN patch significantly reduced the viability of E. coli, S. aureus, and P. aeruginosa, with the 5% GL MN patch exhibiting the strongest antibacterial effect. The patch also showed anti-inflammatory properties, reducing the expression of pro-inflammatory cytokines such as IL-6 and TNF-α. In vivo tests confirmed the safety and efficacy of the MN patch, with no significant toxicity or adverse effects observed. The MN patch provided long-term stability of L. reuteri, with high viability even after 90 days. The study highlights the potential of probiotic-based MN patches as a novel, effective, and safe treatment for chronic wound infections.A probiotic-based microneedle (MN) patch with a 5% glycerol (GL) matrix was developed to accelerate the healing of infected wounds. The MN patch contains Lactobacillus reuteri (L. reuteri), which can metabolize glycerol to produce reuterin, a broad-spectrum antibacterial substance. The MN patch provides a long-acting antibacterial and anti-inflammatory effect, with L. reuteri remaining viable for over 60 days at 4°C. In a mouse model of Staphylococcus aureus-infected wounds, the 5% GL MN patch showed superior antimicrobial efficiency and wound healing performance compared to control groups, indicating its potential for treating chronic infections. The MN patch was fabricated using a vacuum-assisted micromolding method, with different glycerol concentrations tested for optimal performance. The 5% GL MN patch demonstrated efficient delivery of L. reuteri, rapid dissolution after skin insertion, and effective inhibition of bacterial growth. In vitro studies showed that the MN patch significantly reduced the viability of E. coli, S. aureus, and P. aeruginosa, with the 5% GL MN patch exhibiting the strongest antibacterial effect. The patch also showed anti-inflammatory properties, reducing the expression of pro-inflammatory cytokines such as IL-6 and TNF-α. In vivo tests confirmed the safety and efficacy of the MN patch, with no significant toxicity or adverse effects observed. The MN patch provided long-term stability of L. reuteri, with high viability even after 90 days. The study highlights the potential of probiotic-based MN patches as a novel, effective, and safe treatment for chronic wound infections.