Magnesium-doped hydroxyapatite (Mg-HAp) nanofibers show promise for medical applications due to their structural similarity to bone mineral and enhanced biological properties, such as improved biocompatibility and antimicrobial activity. This study synthesized Mg-HAp nanofibers using a microwave-assisted hydrothermal method (MAHM) to evaluate their cytotoxicity, biocompatibility, and antimicrobial efficacy compared to commercial hydroxyapatite (HAp). Characterization through X-ray diffraction (XRD), Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), Energy Dispersive X-ray Spectroscopy (EDS), and Fourier Transform Infrared Spectroscopy (FTIR) confirmed the successful incorporation of magnesium, producing high-purity, crystalline nanofibers with hexagonal morphology. Rietveld refinement showed slight lattice parameter shortening, indicating Mg2+ ion integration. Cell viability assays (MTT and AlamarBlue) revealed a significant increase in fibroblast proliferation with 2% and 5% Mg-HAp concentrations compared to controls (p<0.05), demonstrating non-cytotoxicity and enhanced biocompatibility. Antimicrobial tests (disk diffusion method, 100 µg/ml) showed that Mg-HAp had strong antibacterial effects against Gram-positive and Gram-negative bacteria, and moderate antifungal activity against Candida albicans. In contrast, commercial HAp showed no antimicrobial effects. These results suggest Mg-HAp nanofibers have significant advantages as biomaterials for medical applications, particularly in preventing implant-related infections, supporting further clinical development.