The current research aimed to fabricate ondansetron nanostructured lipid carriers (OND-NLCs) and incorporate them into a suppository base to manage chemotherapy-induced vomiting and nausea, which offer the advantage of both rapid onset and prolonged release. NLCs were fabricated by adopting the solvent diffusion method. The binary lipid mixture of oleic acid (liquid lipid) and lauric acid (solid lipid) were prepared in distinct ratios. The NLCs were characterized concerning the surface charge, size, drug encapsulation efficiency, and surface morphology. In addition, the influence of surfactant, co-surfactant, and lipid on entrapment efficiency and particle size was investigated. Phosphate buffer having pH 7.4 is used for evaluating in vitro drug release by utilizing a dialysis membrane. Various kinetics models were used to estimate the drug release kinetics of fabricated nanostructured lipid carriers. The particle size of the NLCs was calculated between 101 and 378 nm with negative zeta potential on the NLC’s surface. The entrapment efficiency was found between 68 and 87%. Scanning Electron Microscopic analysis showed the spherical shape of nanostructured lipid carriers. The dissolution profile of the ondansetron-loaded NLC suppository depicts biphasic behavior of firstly burst release then slow release was observed. The diffusion controlled release was evident from kinetic modeling. The succeeding step comprehended the fabrication and characterization of NLC-based suppositories utilizing NLC formulations that demonstrated the combined advantage of rapid onset, prolonged release, and better in vivo bioavailability as compared to control suppository.