Green synthesis of metal oxide nanoparticles (NPs) offers an eco-friendly, cost-effective alternative to conventional chemical and physical methods, minimizing energy use and hazardous reagents. This study demonstrates the biogenic production of manganese oxide (MnO) NPs using Catharanthus roseus flower extract as a reducing and capping agent, Comprehensive characterization via FTIR (Mn–O vibrations at 591–405 cm⁻¹ along the capping groups), XRD (confirms the cubic crystalline phase), FESEM (flaky, agglomerated sheets), EDX (Mn 62.37%, O 28.40% and C 9.23%), zeta potential (−0.3 mV), and TGA (33.7% phased mass loss to 985 °C) verified pure and stable MnO NPs. In vitro assays on L929 fibroblasts revealed dose-dependent MTT cytotoxicity (78.77% viability at 20 µg/mL to 39.97% at 100 µg/mL) yet enhanced scratch wound closure (−16.31% area reduction vs. −17.41% control), alongside potent antibacterial activity with highest inhibition zones of 15 mm against Klebsiella pneumoniae and Escherichia coli, and lowest of 4 mm against Pseudomonas aeruginosa at 40–100 µg/mL. These multifaceted properties highlight C. roseus-assisted MnO NPs’ promise for wound healing and antimicrobial applications, warranting dosage optimization and in vivo studies.