We evaluated Sordaria fimicola strains collected from benign and harsh environments of Evolution Canyon 1 (EC 1) for superoxide dismutase (SOD) enzyme activity, and analyzed their respective gene sequences, which were then submitted to the NCBI database for the first time. Ten strains of Aspergillus niger were used as control in a SOD assay. In enzymatic analysis, among 61 isolates the N6 strain of S. fimicola was found to be the most efficient as it caused 50% inhibition of NBT (Nitro-blue tetrazolium) reduction at 20 μg of the SOD protein, while in A. niger, strain 744 showed 60% inhibition of the NBT reduction at 40 μg amount of SOD protein and was found to be most efficient among A. niger. The superoxide dismutase-1 (SOD-1) gene (including exones and introns; 960 bases) was amplified and sequenced from biochemically efficient strains of S. fimicola viz. N6, N7, S2, S1, and SF13, and submitted to the NCBI database under accession numbers KM282180, KM282181, KM282179, KM282178, and KM282177, respectively. On comparison with the reported sequence of Neurospora crassa (M58687.1), a total of 25 base substitutions and seven amino acids changes were detected in the in silico translated proteins of all five strains of S. fimicola compared to the reference sequence of N. crassa (M18334.1). The biochemical as well as molecular data of the study proved that environmental stresses affected the SOD-1 gene by bringing in mutations, which may result in genomic diversity among their frontier molecules such as proteins. The observed enzymatic activity of SOD in S. fimicola strains was reported to be even better and was comparable to A. niger strains, and thus S. fimicola strains can be exploited further for enzymatic production for industrial use.