Biodegradation of chlorpyrifos was studied in mineral medium and soil with a novel fungal strain JAS1 isolated from a paddy field soil. The molecular characterization based on 18S rRNA sequence homology confirmed its identity as Aspergillus terreus. The 300-mg L−1 chlorpyrifos and its major metabolite 3,5,6-trichloro-2-pyridinol (TCP) were completely degraded within 24 h of incubation in the mineral medium. In soil enriched with chlorpyrifos and nutrients (carbon, nitrogen, and phosphorous), A. terreus JAS1 was able to degrade chlorpyrifos and its metabolite TCP (300 mg kg−1 soil) in 24 and 48 h, respectively. The soil was spiked with chlorpyrifos (300 mg kg−1 soil) devoid of nutrients and the fungal strain was capable of degrading both chlorpyrifos and TCP in 24 and 48 h, respectively. The course of the degradation process was studied using high-performance liquid chromatography and Fourier transform infrared analyses. These results showed that the chlorpyrifos-degrading fungal strain had the potential to degrade the pesticide-contaminated agricultural soils even without addition of nutrients.