Cellulose nanofibers, which are troublesome to spin into fibers, can be easily fabricated by post-regeneration of its acetate-derived threads. Cellulose is a natural polymer; it enjoys better biocompatibility, cellular mimicking, and hydrophilic properties than its proportionate analog. Herein, we regenerated acetate-free nanofibers by alkaline de-acetylation of as-spun nanofibers. The resultant cellulose nanofibers previously loaded with hydroxyapatite (HAp) were immobilized using silver (Ag) nanoparticles (NPs) by reduction of adsorbed Ag ions on using sodium borohydride. These amalgamated nanofibers were characterized for SEM, EDX, TEM, FTIR, and hydrophilicity tests revealing the existence of both HAp and Ag NPs in/on the nanofiber scaffolds. The de-acetylation of composite nanofibers resulted in spontaneous hydrophilicity. These nanofibers were cytocompatible, as resolved by MTT assay conducted on chicken embryo fibroblasts. The SEM of the samples after cell culture revealed that these composites allowed a proliferation of the fibroblasts over and within the nanofiber network, and increased concentration of HAp levitated the excessive of apatite formation as well as increased cell growth. The antimicrobial activity of these nanofibers was assessed on E. coli (BL21) and S. aureus, suggesting the potential of de-acetylated nanofibers to restrain bacterial growth. The degradation study for 10, 30, and 60 days indicated degradation of the fibers much is faster in enzymes as compared to degradation in PBS. The results certify that these nanofibers possess enormous potential for soft and hard tissue engineering besides their antimicrobial properties.