The global drive for sustainable food systems highlights the potential of underutilized crops like Moringa stenopetala (MS) for their nutritional and functional properties. This study examines the impact of defatting methods on the physicochemical and functional properties of MS protein isolates prepared using alkaline isoelectric precipitation (ISO) and NaCl membrane filtration (MEM_NaCl). Protein isolates were derived from cold-pressed MS meal, both undefatted (UMGPI) and defatted using acetone (AMGPI), hexane (HMGPI), methanol (MMGPI), ethanol (EMGPI; 70%, 7EMGPI; 50%, 5EMGPI), and water (WMGPI). ISO yielded superior protein recovery (41.73 – 83.23%) and content (69.50 – 83.24%) compared to MEM_NaCl, which achieved lower recovery rates (12.96 – 63.67%) and content (28.21 – 72.83%). Thus, ISO-prepared isolates were prioritized for further analysis. UMGPI provided the highest yield, while AMGPI showed superior protein content and solubility (25 – 75%) across pH levels 3 – 9. MMGPI stood out for high amino acid values, while AMGPI exhibited the highest foaming capacity (~80%) and stability at pH 7 and 9. Protein solubility peaked at pH 3. Intrinsic fluorescence revealed weak tyrosine (303 nm) and pronounced tryptophan peaks (348 nm), with MMGPI showing the highest intensity at pH 3. 7EMGPI excelled in in vitro protein digestibility (81.32%) and oil-holding capacity (3.93 g/g). Water-holding capacity was highest in 5EMGPI (2.81 g/g). UMGPI retained the highest phenolic content (1.39 – 1.53 mg GAE/g), while WMGPI had the lowest (0.57 – 1.29 mg GAE/g). Bitterness intensities ranged from 23.34 (MMGPI) to 28.24 (AMGPI). Enzymatic hydrolysis with alcalase (ALH), flavourzyme (FLH), and pepsin + pancreatin (PPH) improved bioactivity. PPH achieved the highest protein recovery (86.23%), superoxide scavenging (25.77 – 84.55%), and metal chelation (4.64%). ALH demonstrated the highest protein content (72.16%), lowest peptide molecular weight (0.38–2.19 kDa), potent ACE inhibition (15.07 – 66.56%), and renin inhibition (24.89 – 51.68%). FLH excelled in pancreatic lipase inhibition (42.06 – 64.38%) and arginase inhibition (13.18 – 34.57%). These findings highlight the role of defatting methods in optimizing MS protein functionality, with AMGPI emerging as a promising candidate for functional foods and nutraceuticals. Enzymatic hydrolysis, particularly with alcalase, further broadens their application potential.