Thermotolerant microorganisms were collected, identified and characterized under different physiological conditions from various rotten fruits in Bangladesh for vinegar production. Among the 15-isolates characterized previously, the strains F-1, F-3 and F-10 represented Staphylococcus, Bacillus and Acetobacter spp., respectively. After checking various parameters for growth, acetic acid production rate was optimized further. Among the 3-starins analyzed here, the strain F-10 gave maximum acetic acid (7.0 g/100 ml) at 37˚C in 2% ethanol concentration. The strain F-10 is capable of producing high yield of acetic acid at relatively high temperature, which is an ideal condition for vinegar production, which may reduce the water cooling expenses as well as the risk of contamination.