The biodegradation of profenofos, an organophosphorus insecticide, by four Bacillus subtilis strains, namely, DR-39, CS-126, TL-171, and TS-204, isolated from grapevines or grape rhizosphere was studied in liquid culture, on grape berries, and in vineyard soil. Each of the four B. subtilis strains enhanced the degradation of profenofos in all three matrices. Degradation rate constants were best obtained by first + first-order kinetics module. In nutrient broth spiked with 5 μg/mL profenofos, inoculation with B. subtilis strains DR-39, CS-126, TL-171, and TS-204 reduced the half-life (DT50) of profenofos to 4.03, 3.57, 2.87, and 2.53 days, respectively, from the DT50 = 12.90 days observed in the uninoculated control. In Thompson Seedless grapes sprayed with profenofos at a field dose of 1250 mL ai/ha, the DT50 values were 1.07, 1.00, 2.13, and 2.20 days in grapes inoculated with B. subtilis strains DR-39, CS-126, TL-171, and TS-204, respectively, as compared to 2.20 days in uninoculated grapes. These four B. subtilis strains also enhanced the degradation of profenofos in autoclaved soil (DT50 = 5.93, 7.47, 6.00, and 4.37 days) and in nonautoclaved soil (DT50 = 0.87, 2.00, 2.07, and 2.43 days) amended with 5 μg/g profenofos from the half-lives of 17.37 and 14.37 days in respective uninoculated soils. Growth dynamic studies indicated that all four B. subtilis strains were able to establish and proliferate on berries and soil equally well in the presence or absence of profenofos. Degradation product 4-bromo-2-chlorophenol was identified by GC-MS. Strain DR-39 was most effective in the natural environments of grape and soil.