The alarming health issues especially the unusually high number of cancer cases in agriculture community of Bathinda district of Punjab (India) is a serious concern. There is limited knowledge about the role of gene-environment interactions in oncogenesis prevalent in this area. The aim of this study was to evaluate the association of oxidative stress with CYP1A2, CYP2B6, CYP2C9, CYP3A4, and PON1 genetic variation in the pesticide-exposed (occupationally) population of Bathinda district of Punjab (India). This study demonstrated significantly elevated relative risk (RR) of lower antioxidant defense mechanism (Glutathione, Catalase, Superoxide Dismutase, Glutathione peroxidases, and Glutathione Reductase) in occupationally pesticide-exposed group (n = 120) as compared with unexposed group (n = 84) from Bathinda district of Punjab (India). Our data shows pesticide exposure to be a major risk factor leading to increased oxidative stress inside the body. Gas chromatographic analysis revealed the residues of organophosphates (chlorpyriphos, dichlorvos, ethoprophos) and herbicides (atrazine, butachlor, alachlor, metolachlor) in the blood samples of the exposed population. In vitro results showed a dose dependent decrease in cell viability following treatment of pesticides detected in blood samples in hPBMCs and A549 cell line. Genetic variation analysis revealed missense mutations in CYP2B6 (2 mutations), CY3A4 (1 mutation), and CYP2C9 (2 mutations). The observed mutations have been predicted to cause structural and conformation change in protein structure which could result in altered stability. In first of its kind of study, our data reveal oxidative stress and pesticide residue accumulation inside the body to be the major reasons for health concerns in Bathinda district.