Poly(lactic acid) (PLA)/cellulose nanocrystal (CNC) composite scaffolds were fabricated using an electrospinning technique to evaluate the influence of CNCs on the biocompatibility and osteogenic potential of PLA. The scaffolds were characterized using Fourier transform infrared spectroscopy (FTIR), X-ray diffraction pattern (XRD), transmission electron microscopy (TEM), and atomic force microscopy (AFM). A significant enhancement of the mechanical properties occurred in the composite scaffolds compared to pure polymer. This is due to the stronger interactions between the polymer chains and CNCs. The composite scaffolds exhibited higher thermal stability compared to pure polymer. Notably, excellent adhesion and proliferation was observed in the presence of the fabricated composite scaffolds, indicating their superior biocompatibility. Higher mineralization was noted on the surface of composite scaffolds. The fabricated scaffolds were significantly covered by the cultured cells and exhibited greater fluorescence intensity vis-à-vis control. Additionally, the fact that higher expression of osteogenic gene markers was observed in composite scaffolds confirms their enhanced osteogenic potential. The bone regeneration potential of the fabricated scaffold was monitored in a rat calvarial defect model after 3 weeks of treatment. The fabricated scaffold demonstrated excellent biocompatibility and superior osteoinductivity. Therefore, the fabricated scaffolds possess potential to be used as a biomaterial for tissue engineering applications.