Background: Freshwater microalgae are a promising sustainable resource for biomass production, biofuel development, and nutraceutical applications. Pakistan's diverse freshwater ecosystems remain largely unexplored for indigenous algal species with commercial potential.

Methods: Six algal species (Cosmerium gemmiferum, Sunedesmus oblicuus, Desmodesmus maximus, Chlorella pyrenoidosa, Oocystis lacustric, and Odogonium cardiacum) were isolated from five freshwater locations in Malakand region, Khyber Pakhtunkhwa, Pakistan. Biomass production was optimised using BBM in controlled bioreactor systems. Comprehensive physicochemical characterisation included proximate analysis (AOAC methods), mineral profiling (AAS, flame photometry, and spectrophotometry), and statistical analysis using two-way ANOVA.

Results: C. gemmiferum demonstrated superior biomass productivity (0.52±0.05 g/L) and the highest carbohydrate content (NFE: 71.35±2.8%). Significant interspecies variation was observed in the proximate composition, with crude protein ranging from 0.25-0.47%, crude fat 3-19%, and ash content 8-54%. Mineral analysis revealed species-specific accumulation patterns: O. cardiacum showed maximum ash (51.6±3.2%) and potassium content (8.44±0.6 mg/kg), while C. gemmiferum exhibited highest iron (1.33±0.15 mg/kg) and zinc (0.94±0.08 mg/kg) concentrations. ANOVA revealed highly significant species effects (p<0.0001) for most parameters, with location showing a variable influence.

Conclusions: Indigenous freshwater algae from the Malakand region have substantial biotechnological potential for sustainable biomass production. C. gemmiferum emerges as a promising candidate for biofuel applications due to high carbohydrate content and biomass yield, while O. cardiacum shows potential for mineral-rich nutraceutical applications. These findings provide a foundation for developing region-specific algal cultivation strategies in Pakistan's transition toward a sustainable bioeconomy.