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Traditional fermentation using natural microorganisms often results in uncontrolled and inconsistent product quality. Identifying dominant LAB and yeast species through morphological, physiological, and biochemical characterization, this study aimed to optimize the traditional fermentation process of injera dough fermentation. Ersho and dough were prepared in the laboratory, and LAB and yeast were identified and enumerated by smearing on a petri dish at 24-hour intervals from 0 to 96 hours of fermentation. The growth kinetics of both LAB and yeast in fermented teff dough significantly increased (p < 0.05) from 8.91 to 9.97 log CFU/gm and 5.98 to 7.62 log CFU/gm as fermentation time increased. Five dominant LAB species (Lactobacillus fermentum, Lactobacillus Brevis, Lactobacillus Plantarum, Bacillus subtilis, and Enterococcus casseliflavus) and three dominant yeast strains (Saccharomyces cerevisiae, Candida krusei, and Pichia kudriavzevii) were identified. Based on the physicochemical analysis and sensory acceptability result, application of a single starter culture in teff dough fermentation improved fermentation time and acid production. [ABSTRACT FROM AUTHOR]