Agricultural pesticides are one of the indispensable materials used to meet increasing demand of food crops. Consequently, pesticides find their way to various ecosystems resulting in detrimental pollution problems. Chlorpyrifos is a predominantly used organophosphorus pesticide leading to harmful effects including abnormal cell division. Scientific attempt to eliminate these contaminants from the environment using biodegradation approach has been appreciated. Soil bacteria capable of utilizing chlorpyrifos are potential bioremediation candidates. In context to the same, the present study isolated six bacteria growing in presence of chlorpyrifos as sole carbon source from agricultural soils. The obtained bacterial isolates characterized based on their morphological characteristics, biochemical reactions were found to be Pseudomonas spp. JR16, Pseudomonas spp. J1, Pseudomonas spp. J2, Pantoea spp. J3, Enterobacter spp. J4 and Kocuria spp. J5. Among these, the most potential of them viz., Pseudomonas spp. JR16, was further proceeded for molecular identification and was observed to be the Pseudomonas putida JR16. The influence of different chlorpyrifos concentration, various carbon sources, metals sources, temperature and pH on the growth of Pseudomonas putida JR16 was assessed for developing optimum conditions for on field application of the strain for biodegradation of chlorpyrifos.