Oxidative Stress is an imbalance between the systemic manifestation of reactive oxygen species and a biological system's ability to repair the resulting damage that leads to deleterious effects in living organisms. Stress has been reported to be a causative factor for male infertility. Withania somnifera has been documented in Ayurveda for its stress-combating properties. However, limited scientific literature is available on aspect of W. somnifera grown in different soil at different regions. We undertook the present study to understand the role of stress in male infertility, and to test the ability of W. somnifera grown in black soil and red soil to combat stress and treat male infertility. Plants containing high amount of antioxidants that protect the body cells against stress. Antioxidants have the ability to scavenge and neutralize the generated free radicals. In the present study we evaluated the antioxidant potential of ethanolic root extracts of Withania somnifera grown in black soil (BWS) and Withania somnifera grown in red soil (RWS) in Drosophila melanogaster under oxidative stress state. To know about the effect on male fertility, sperm count and accessory gland protein estimation were carried out in extract fed flies. The efficacy of Withania somnifera in scavenging reactive oxygen species was measured through biochemical analysis. The total sperm count and ACp amount was more in BWS and in OS induced BWS fed flies. Further, it reveals that BWS extract fed flies increases the SOD activity by 11.27%, CAT activity by 7.13%, GSH increases by 7.34% when compared to control group. The activity levels of antioxidant enzymes in Paraquat induced BWS extract fed batches showed significant increase in SOD (18.23%), CAT (11.29%) and GSH (14.17%) when compared to control flies with the induction of OS. Lipid peroxidation also decreased in BWS fed flies under stress (70.13%) and non-stress conditions (91.14%). This was due to high degree of antioxidants and natural polyphenols in Withania somnifera grown in black soil region.