Abstract : The objective of the study was to assess the conservative effects of L-Ergothioneine (EGT), antioxidant, on the post thaw chromatin integrity of human spermatozoa. 400 normozoospermic semen samples were cryopreserved with (Test) and without (Control) supplementation of EGT. Clinically relevant tests such as Semen analysis (WHO 2010), Sperm chromatin integrity (Sperm chromatin dispersion test) and Lipid peroxidation status (Malondialdehyde concentration assessment) were performed in both fresh and frozen thawed samples. It was seen that with the increase in ROS production due to cryopreservation, there is a significant decrease in the post thaw sperm parameters and sperm chromatin integrity (Fresh vs Control: p<0.001), the extent of which was reduced on addition of EGT (Test vs Control: p<0.001). Also interestingly, the increasing ROS levels (assessed in MDA test) correlated directly with DNA damage. Hence the study provides a basal proof that EGT acts against the ROS and conserves the spermatozoa's functionality.