Cost effective bioethanol production from biomass requires complete utilization of mixed sugars and their efficient fermentation to ethanol. The fermenting strain should be capable of hexose and pentose utilization and tolerant to inhibitory byproducts of pretreatment. While, metabolic engineering strategies on Saccharomyces have yielded laboratory strains capable of mix sugar fermentation, hardly a few approaches have realized in industrial strains. Therefore, bioprospecting non-conventional native yeasts for efficient utilization of carbohydrate component of the biomass is imperative. In the present study, the fermentation efficiency of naturally pentose utilising yeasts such as Pichia stipitis NCIM3498, Pichia stipitis NCIM3497, Candida tropicalis Y6 and Rhodotorula glutinis Y1 was assessed on alkali pretreated rice straw hydrolysates, synthetic sugar/mixture and in presence of inhibitors. Highest fermentation efficiency (57.30%) on hydrolysate within 24 h was observed in P. stipitis NCIM3497 while P. stipitis NCIM3498 and C. tropicalis Y6 showed 53.03 and 46.51% respectively. On 2% glucose, fermentation efficiency was 64.77%–86.96% for Pichia and Candida strains, complete sugar depletion with 8.87 g L−1 highest ethanol production. On mixed sugars, highest fermentation efficiency was 87.35%. Pichia and Candida strains were tolerant to furfural and produced ethanol. Acetic acid and formic acid inhibited growth, sugar consumption and no ethanol detected. Display omitted •Pichia stipitis, Candida tropicalis, Rhodotorula glutinis grew on straw hydrolysate.•P. stipitis NCIM3498 consumed >90% sugars producing 8.8 gL-1 ethanol on hydrolysate.•C. tropicalis Y6 used 57% sugars producing 2.84 gL-1 ethanol on straw hydrolysate.•P. stipitis NCIM3498 produced ∼4% ethanol in optimized synthetic mixed sugar medium.•Yeast strains were tolerant to furfural but inhibited by formic and acetic acid.