The impact of saturation and unsaturation in the fatty acyl hydrocarbon chain on the physicochemical properties of nanostructured lipid carriers (NLCs) was investigated to develop novel delivery systems loaded with an anticancer drug, ursolic acid (UA). Aqueous NLC dispersions were prepared by a high-pressure homogenization-ultrasonication technique with Tween 80 as a stabilizer. Mutual miscibility of the components at the air-water interface was assessed by surface pressure-area measurements, where attractive interactions were recorded between the lipid mixtures and UA, irrespective of the extent of saturation or unsaturation in fatty acyl chains. NLCs were characterized by combined dynamic light scattering, transmission electron microscopy (TEM), atomic force microscopy (AFM), differential scanning calorimetry, drug encapsulation efficiency, drug payload, in vitro drug release, and in vitro cytotoxicity studies. The saturated lipid-based NLCs were larger than unsaturated lipids. TEM and AFM images revealed the spherical and smooth surface morphology of NLCs. The encapsulation efficiency and drug payload were higher for unsaturated lipid blends. In vitro release studies indicate that the nature of the lipid matrix affects both the rate and release pattern. All UA-loaded formulations exhibited superior anticancer activity compared to that of free UA against human leukemic cell line K562 and melanoma cell line B16.