The traditional fermentation of Ethiopian injera, using the natural mixed starter culture Ersho, often results in extended fermentation times and inconsistent product quality. This study aim to address these challenges by isolating and molecularly characterizing the dominant LAB and yeast species involved in teff injera dough fermentation. Therefore, the study was conducted to investigate the effect of mixed microbial starter culture and fermentation time on physicochemical, sensory and microbial quality of injera. Treatments include L1Y1, L1Y2, L2Y1, and L2Y2, each with 1 mL equal inoculum, fermented for 18, 30, and 42 h. The experiment was conducted in a CRD with factorial arrangements (5×3=39 treatments) with triplicates. Standard methods were used to isolate and characterized the microbes, and to conduct physicochemical, sensory and microbial quality analysis of injera. Phenotypically four dominant LAB species were isolated: Lactobacillus fermentum (31.25%), Lactobacillus plantarum (37.5%), Bacillus subtilis (18.75%), and Enterococcus casseliflavus (12.5%). Three yeast species were identified: Saccharomyces cerevisiae (44.44%), Pichia kudriavsevii (33.33%), and Candida krusei (22.22%). Molecular identification confirmed L. plantarum (23.5%) and L. fermentum (29.41%) as the primary LAB species, and Saccharomyces cerevisiae (7.69%) and Pichia kudriavsevii (23.08%) as the main yeast species. MALDI-TOF MS further identified LABs (11.76%) as L. plantarum and fermentum, and yeasts (84.61%) as Candida kruse. Significant differences (p≤0.05) in pH and TA were observed in fermented teff dough. The microbial load of injera ranged from 2.07 to 2.56 log CFU/g and 2.43 to 3.96 log CFU/g (aerobic plate count), and 3.44 to 5.12 log CFU/g and 4.52 to 6.04 log CFU/g (yeast and mold) for the 3rd and 5th day of storage, respectively. Sensory evaluation revealed panelists preferred injera fermented with L2Y1 at 30 h for its color, texture, aroma, and rollability. The LlY2 at 18 h fermentation was favored for top and bottom surface, eye uniformity, and overall acceptability. L2Y2 at 30 h fermentation was preferred for taste. The application of mixed starter cultures decreased fermentation time and increased acid production rapidly. Further studies on molecular characterization of LAB and yeast using 16S rRNA sequencing are recommended to confirm the isolates. Keywords: Injera quality, LAB, microbial fermentation, mixed starter culture, molecular characterization, teff dough, yeast,