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Abstract Fermentation of cereal-made dough was the primary Ethiopian food for many years. Sourdough is believed to have many beneficial fermenting micro-floras depending on the source of dough and environmental conditions. The study aimed to isolate and characterize yeasts from fermenting cereal dough and evaluate their leavening potential for wheat dough. Morphological and biochemical characterizations such as sugar fermentation, glucose tolerance test, growth at different pH and temperatures, and agitation effect were employed to select potent yeasts. Accordingly, 16 potential yeast isolates were selected and subjected to further analysis. Finally, the yeasts were tested and compared for their leavening activity on the wheat dough at 37 °C and room temperature. Results revealed marvelous CO2 producer mesophile yeasts that degrade various sugars and tolerate up to 60% glucose concentration. The overall characterization assigned all the yeast isolates to the genus Saccharomyces and the Candida humilis. ANOVA showed that leavening activity by the yeast isolates was statistically higher at 37 °C compared to room temperature. The highest dough volume (6.2 mL) was recorded from Wh17, Wh5, and Ba12 isolates. Sor9 isolates were among the yeast that raised a minimum dough volume (0.7 mL). In contrast to commercial yeasts, the present study detected exceptional yeasts that matured within 24 h. Therefore, the discovered potential yeasts can be used for leavening dough in a short fermentation time and add aroma to baked products, making them a candidate in the baking industry. It could also be cost-effective as it could reduce media consumption and electricity utilization.