The imperative of utilising alternative fuels for the operation of internal combustion engines stems from the requirements to reduce the emissions of greenhouse gases and other contaminants, the substantial demand for fuels, and the diminishing reserves of natural resources. The global inclination towards sustainable development necessitates the employment of biofuels as a substitute for fossil fuels. Nonetheless, the expenditures on raw materials for the manufacture of biodiesel remain substantial, thus underlining the importance of exploring solutions for reducing them. An instance of this could be the utilisation of plant and animal by-products, such as used frying oils and slaughterhouse waste, as feedstock for biodiesel production. Not only will this facilitate the creation of less costly biofuel, but it will also provide an effective solution for the management of post-production waste. The objective of the research delineated in this paper was to ascertain select physicochemical attributes of second-generation biodiesel, derived from spent frying oil, as well as mixtures of this biodiesel with diesel and biodiesel concentrations of 10, 20, and 30% (v/v). The biodiesel produced is the waste frying oil methyl esters WFOME. The proprietary GW-201 reactor was employed in the production of biodiesel. For WFOME biodiesel, DF diesel, and their blends—B10, B20, and B30—properties that influence the formation process of the combustible mixture, autoignition, and combustion of fuel–air mixtures in self-ignition engines were determined. The conducted research has established that “B” type fuels prepared from WFOME and DF present a viable alternative to fossil fuels. Pure biodiesel exhibited a marginally reduced lower heating value, however, in the case of fuel mixtures comprising up to 30% (v/v) biodiesel and diesel, the lower heating values approximated that of diesel. An elevated cetane number alongside an increased flash point of pure B100 biodiesel have been noted. The values of cetane number for WFOME and DF mixtures were found to be either comparable or marginally higher than those of pure DF diesel fuel.