A bacterial strain that utilized o-chloronitrobenzene (o-CNB) as the sole carbon, nitrogen and energy sources was isolated from an activated sludge collected from an industrial waste treatment plant. It was identified as Pseudomonas putida based on its morphology, physiological, and biochemical characteristics with an automatic biometrical system and the 16S rRNA sequence analysis. Microcosm study showed that the biodegradation of o-CNB was optimized at culture medium pH 8.0 and temperature of 32℃. At these conditions, the strain degraded 85% of o-CNB at a starting concentration of 1.1 mmol/L in 42 h. o-Chloroaniline was identified as the major metabolite with both high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). The study showed that o-chloronitrobenzene degradation by Pseudomonas putida OCNB-1 was initiated by aniline dioxyenase, nitrobenzene reductase and catechol-1,2-dioxygenase.