<p>Full-thickness wound healing, particularly in chronic wounds, has remained a significant clinical challenge due to limited regenerative therapies and immune responses. Although the different biologic-based combination therapies have been suggested, the effective and safe biological adjuvants require further research and development to optimize their potential for wound healing. This study investigates the synergistic effect of menstrual blood mesenchymal stem cell-derived extracellular vesicles (MenMSC-Evs) combined with platelet-rich fibrin (PRF) on full-thickness wound healing. In this context, the MenMSC-Evs and PRF were first assessed in vitro (<i>n</i> = 3) for their antibacterial efficacy against E. coli, S. aureus, and P. aeruginosa. The impact of MenMSC-Evs on the proliferation and migration of HUVECs and NIH3T3 cells was evaluated to determine their influence on cellular behaviors. In vivo, a full-thickness excisional wound model was established on nine Wistar rats, with four wounds per rat assigned to different treatment groups, to evaluate wound healing. In vitro, MenMSC-Evs significantly enhanced the proliferation and migration of HUVECs by 29.3% and 11.5%, respectively, and NIH3T3 cells by 24.7% and 8.5% (<i>p</i> &lt; 0.01, <i>p</i> &lt; 0.001). PRF-Evs also demonstrated improved antibacterial activity, particularly against S. aureus (<i>p</i> &lt; 0.001). In vivo, PRF-Evs accelerated wound closure by 23.2% (<i>p</i> &lt; 0.001), increased collagen deposition (<i>p</i> &lt; 0.001), and promoted angiogenesis, as evidenced by a 1.9-fold increase in VEGF-A expression and a 3.1-fold rise in CD34-positive microvessel density (<i>p</i> &lt; 0.001). Moreover, PRF-Evs modulated macrophage polarization by reducing pro-inflammatory cytokines—IL-1β (8.2-fold) and TNF-α (4.4-fold)—while upregulating anti-inflammatory cytokines, including IL-10 (3.2-fold) and TGF-β (2.7-fold) (<i>p</i> &lt; 0.001). In conclusion, these findings indicate that the novel combination of MenMSC-Evs and PRF offers a promising cell-free therapeutic strategy for chronic wound management by enhancing immunomodulation, promoting neovascularization, and facilitating tissue regeneration.</p> Graphical Abstract <p>Schematic overview of Evs isolation from MenMSCs and integration with PRF for wound healing. <b>a</b>) PRF was extracted from venous blood and subsequently freeze-dried. <b>b</b>) Evs were extracted from MenMSCs and seeded onto a PRF. <b>c) </b>Characterization of the Evs was performed using DLS, TEM, flow cytometry, and protein assays, while the PRF-Evs complex was analyzed via FE-SEM. <b>d</b>) In vitro assays evaluated the effects of MenMSC-Evs on the proliferation and viability of HUVEC and NIH3T3 fibroblast cell lines, alongside antibacterial assessments of PRF, MenMSC-Evs, and PRF-Evs complexes against E. coli, S. aureus, and P. aeruginosa. <b>e</b>)The therapeutic efficacy of PRF, MenMSC-Evs, and PRF-Evs complexes on wound healing in Wistar rats was analyzed through metrics including wound closure dynamics, histopathological evaluations (H&amp;E staining), angiogenesis (CD34 and VEGF-A), collagen deposition (Masson's trichrome staining, collagen I, and collagen III assessments), and macrophage polarization (CD68 expression and cytokines assay). (Abbreviations: MenMSCs: menstrual-derived mesenchymal stem cells, PRF: platelet-rich fibrin, PRF-Evs, platelet-rich fibrin loaded with Evs, DLS: dynamic light scattering, TEM: transmission electron microscopy, FE-SEM: field emission scanning electron microscopy, HUVEC: human umbilical vein endothelial cells)</p> <p></p>