The increasing demand of energy has been supplied through the combustion of petroleum throughout the world. Due to escalating cost of petroleum, its contribution to global warming and non-renewable nature of this oil, there is collectively a need for renewable and ecofriendly energy sources such as bioethanol. Thus, the objective of this study was aimed at isolating, characterizing and evaluating the potent wild yeasts under different stress conditions from locally available resources for bioethanol production to minimize the utility of fossil fuel. In this study, a total of 35 samples of sugary substrates were collected from Metehara Sugar Factory for wild yeasts isolation following the standard protocols. A total of 305 yeast isolates were retrieved and screened using physiological and osmotic stress tolerance tests. Fermentative and potent wild yeasts were identified to species level using morphological and biolog-based biochemical methods. Out of 305 yeast isolates, 20 (6.56%) and 7 (2.29%) of them were found to be tolerant to 18 and 20% of ethanol, respectively. Out of these 20 ethanol tolerant yeast isolates, 17 tolerated the temperature of 45Â°C for 48 hrs. From 17 ethanol-thermotolerant isolates, 5 (29.41%), 5 (29.41%) and 7 (41.18%) were found producing gas from glucose at 24, 48 and 72 hrs, respectively. Out of the 17 ethanol-thermotolerant yeast isolates, 12 yeast isolates were able to tolerate 35% of glucose. Out of these 12 ethanol-thermo-and-sugar tolerant yeasts, the 7 were found tolerant to pH 2. From the 7 acidic tolerant yeast isolates, 5 yeast isolates were shown tolerant to 7% of NaCl and identified as K. lodderae, P. guilliemodii B, S. boulardii, Z. rouxii and T. globosa, respectively. During fermentation, K. lodderae, P. guilliemodii B, S. boulardii, Z. rouxii and T. globosa were able to produce bioethanol with the values of (% v/v) 12.62, 11.61, 10.58, 10.82 and 10.44, respectively at 48 hrs and 12.56, 12.52, 12.08, 11.11 and 11.48, respectively at 72 hrs. The initial inoculum cell density was increased from (1.83 to 3.08, 0.59 to 1.81, 0.54 to 1.36, 0.48 to 1.69 and 0.45 to 1.56) x108 cells/ml for K. lodderae, P. guilliemodii B, S. boulardii, Z. rouxii and T. globosa, respectively 0 hr to72 hrs. The fermentation efficiency (%) for K. lodderae, P. guilliemodii B, S. boulardii, T. globosa and Z. rouxii was shown as 98.0, 97.8, 93.1, 90.8 and 88.1, respectively. Thus, on the basis of highest stress tolerant and higher fermentation efficiency features, K. lodderae and P. guilliemodii B wild yeasts were considered to be the best bioethanol producers. Molecular characterization and optimization of fermentation parameters is recommended to utilized these potent yeasts.