This study presents a novel probiotic-based microneedle (MN) patch designed to accelerate the healing of infected wounds. The patch, containing *Lactobacillus reuteri* (L. reuteri), is loaded with 5% glycerol and delivered through MNs to the wound site. L. reuteri continuously produces antimicrobial substances, particularly reuterin, by metabolizing glycerol, which helps eliminate harmful bacteria and reduce inflammation. The MNs are made of polyvinyl alcohol (PVA) and sucrose, providing mechanical strength and rapid dissolution upon skin insertion. In vitro studies showed that the 5% GL MN patch effectively inhibited the growth of common wound bacteria, such as *Staphylococcus aureus*, *Escherichia coli*, and *Pseudomonas aeruginosa*, and promoted wound healing in a mouse model of *S. aureus*-infected wounds. The patch exhibited superior antimicrobial efficiency and accelerated wound closure compared to control groups, demonstrating its potential for treating chronically infected wounds. The MN patch's biocompatibility and stability were also evaluated, showing no significant cytotoxicity and long-term viability of L. reuteri inside the MNs. Overall, this study highlights the promising therapeutic potential of the probiotic-based MN patch for managing infected wounds.This study presents a novel probiotic-based microneedle (MN) patch designed to accelerate the healing of infected wounds. The patch, containing *Lactobacillus reuteri* (L. reuteri), is loaded with 5% glycerol and delivered through MNs to the wound site. L. reuteri continuously produces antimicrobial substances, particularly reuterin, by metabolizing glycerol, which helps eliminate harmful bacteria and reduce inflammation. The MNs are made of polyvinyl alcohol (PVA) and sucrose, providing mechanical strength and rapid dissolution upon skin insertion. In vitro studies showed that the 5% GL MN patch effectively inhibited the growth of common wound bacteria, such as *Staphylococcus aureus*, *Escherichia coli*, and *Pseudomonas aeruginosa*, and promoted wound healing in a mouse model of *S. aureus*-infected wounds. The patch exhibited superior antimicrobial efficiency and accelerated wound closure compared to control groups, demonstrating its potential for treating chronically infected wounds. The MN patch's biocompatibility and stability were also evaluated, showing no significant cytotoxicity and long-term viability of L. reuteri inside the MNs. Overall, this study highlights the promising therapeutic potential of the probiotic-based MN patch for managing infected wounds.