A wide diversity of Nepalese native flora can serve as source of microorganisms (especially yeast) that can be efficiently used as Baker’s yeast. This study focused on the isolation of yeast strains with leavening capabilities from locally available sources like fruits, soil, murcha (traditional starter for fermentation) and a local tree named ‘Dar’ (Boehmeria rugulosa). A total of 10 samples were collected randomly from different localities of Bhaktapur and Kavre districts of Nepal. Dar bark was collected from local forest of Syangja. Following enrichment and fermentation of the samples, 30 yeast strains were isolated using selective medium Wallerstein Laboratory Nutrient Agar. Differential tests were performed which included morphological and microscopic observation, physiological and biochemical characterization that helped for the identification of yeasts. From the 30 isolated strains, 13 were selected as Saccharomyces cerevisiae by nitrate reduction and lactose utilization tests. The selected strains were further assessed by several tests for their efficient leavening ability and the 5 most potent strains FAPW, ENG, MUR3B, SUG1 and DPSG1 isolated from Apple, Grape, Murcha, Sugarcane and Dar respectively, were used in the fermentation and baking of dough and they showed comparable performance than the commercial strain of baker’s yeast.