Chlorpyrifos, an organophosphate (OP) pesticide, possess broad-spectrum insecticidal activity against Lepidoptera, Diptera, Homoptera, Coleoptera, Orthoptera, Hymenoptera, and Hemiptera. Organophosphate pesticides have acute neurotoxicity because they suppress acetylcholine esterase (AChE) which regulates the enzyme of neurotransmitters by reducing acetylcholine concentration at synaptic junctions. On exposure to OP, AChE is inactivated and leads to accumulation of acetylcholine in the junction. Moreover, due to their persistence nature they biomagnify at different food chain levels. In the present study, a newly isolated indigenous bacterial strain Tistrella sp. AUC10 was exploited for its ability to degrade chlorpyrifos pesticide. The isolate completely degrades (99.86%) chlorpyrifos. The degraded metabolites of chlorpyrifos by Tistrella sp. AUC10 were identified by GC-MS analysis. Based on the GC-MS analysis, a putative pathway of degradation of chlorpyrifos by Tistrella sp. AUC10 has been proposed. It emulsified various hydrocarbons and also confirms biosurfactant production on CTAB agar. This study is the first report on elucidation of the mechanism of degradation of chlorpyrifos by Tistrella sp. Furthermore, phytotoxicity studies of the parent compound and the biodegraded chlorpyrifos products revealed drastic reduction in the toxicity of metabolites as compared to the parent chlorpyrifos. This implies that the biodegraded metabolites of chlorpyrifos are of non-toxic nature. This study thus indicates the efficacy of Tistrella sp. AUC10 for the treatment of chlorpyrifos contaminated agricultural fields.