High costs and ethical issues have prompted research into novel non-food feedstocks for the fermentation-based manufacture of sustainable fuels. In this study, Prosopis africana pods (PAP), an underutilized substrate, was examined for its ability to produce bioethanol. The biomass was pretreated with four mushrooms to delignify it and enhance hydrolysis. A scanning electron microscopy (SEM) was performed on raw and pretreated PAP. The optimum hydrolysis conditions for the pretreated biomass were then determined using the Design of Experiment (DOE) approach. The acid type (HNO3 and H3PO4), concentration (1 %, 3 % and 5 %), solid loading (SL; 5 %, 10 % and 20 %) and contact time (15, 30 and 60 minutes) were optimized using a full-factorial design. The most tolerant yeast isolate from different sources was then molecularly identified after being tested for ethanol tolerance. A half-factorial design was used to screen the fermentation factors, and the Box-Behnken design was used to optimize the relevant components. Ganoderma lucidum showed the most luxurious growth during PAP pretreatment and SEM revealed reduction in biomass crystallinity. The hydrolysis conditions of 5 % HNO3, 20 % SL and 15 minutes contact time were optimal, producing 43.37 ± 0.35 g/L of reducing sugars. The most ethanol-tolerant strain, identified as Pichia kudriavzevii SY4, produced 38.26 g/L bioethanol concentration after RSM optimisation. Similarly, optimisation raised bioethanol concentrations from 26.62 ± 0.00 to 38.26 ± 0.18 g/L, a 43.73 % increase. This work is the first report on utilising Prosopis africana pods in bioethanol production.