The acetone-butanol-ethanol as a source of liquid fuel from fermentation process using isolates of the anaerobic bacterium Clostridium sp. continues to attract the attention for replacement of conventional liquid fuels. This study aims at isolation and characterization of native anaerobic clostridia from different biodiversity zones of North Karnataka and detection of presence of organic acids by HPLC analysis. As a result, eighty mesophilic, spores forming, anaerobic, butanol producing Clostridium isolates were isolated. Based on the morphological, biochemical and physiological characterization, 21 Clostridium isolates belonged to the group of clostridia namely C. acetobutylicum, C. beijerinckii and Clostridium saccharobutylicum. The acidogenic strain tentatively identified as C. acetobutylicum [MR-10-3(3)] showed maximum production of butyric (121.88 mg/ml), acetic acid (101.40 mg/ml) and least propionic acid (21.01mg/ml) compared to the reference strain Clostridium acetobutylicum ATCC 824 (29.62 mg/ml of butyric acid, 33.79 mg/ml of acetic acid and 271.62 mg/ml of propionic acid).