Abstract: Background and Objective: The growing disquiets about the dearth of remnant fuels, the release of green house gasses and air pollution by incomplete incineration of fossil fuel have also resulted in an increasing focus on the use of cellulases to perform enzymatic hydrolysis of the lignocellulosic materials for the generation of bioethanol. The aim of this study was to isolate a potential thermotolerant cellulase producing bacterium from natural resources for the bioethanol production. Materials and Methods: The soil samples were collected aseptically from different site of university campus to isolate cellulase producing bacteria by serial dilution method on CMC agar plates (pH 7.0) at 55°C. Fifty bacterial strains isolated from the soil samples were screened for cellulase production. Among them, two cultures were adjudged as the best cellulase producer and were identified as Streptococcus and Bacillus sp. on the basis of biochemical characterization. After growth, incubated plates were overlaid with congo-red solution (0.1%) for 10 min and then washed with 1 N NaOH solution for de-staining and effective cellulolytic bacterial culture were screened out on the basis of clear zone around the colony for further study. The selected bacterial isolates were identified from MTCC Chandigarh. Data were analyzed by one way ANOVA. Results: The Bacillus sp. showed maximum cellulase production (170 U mL–1) in the presence of sugarcane baggase, ammonium sulphate and Mn2+ ions at 55°C, pH 7.0 within 72 h of incubation while Streptococcus sp. showed maximum cellulase production (130 U mL–1) in the presence of wheat bran, ammonium sulphate and K2+ ions at 50°C, pH 6.5 within 96 h. The enzyme showed maximum activity in the presence of Triton-X-100. Tween-40, Tween-60 and Tween-80 (100 mM), was also found to stimulatory effect, respectively. Conclusion: These isolates (Bacillus and Streptococcus sp.) may be useful in several industrial applications owing to its thermotolerant, heavy metal resistant and surfactant resistance characteristics.