Antioxidant enzymes such as glutathione S-transferases (GSTs) and cytochromes P450 (CYPs) are involved in the metabolism and detoxification of cytotoxic compounds, as well as the elimination of reactive oxygen species (ROS). Therefore, alterations in the structure of these enzymes could result in prolonged production of ROS with subsequent risk of development of disorders such as presbycusis. This study aimed to investigate the association between CYP1A1 (rs4646903, rs1048943) and GSTs (GSTM1-deletion, GSTT1-deletion, GSTP1-rs1695) with presbycusis risk in an Iranian population which was followed by an in silico approach. In a case-control study, 280 subjects including 140 cases with presbycusis and 140 healthy controls were enrolled. Genotypes of single-nucleotide polymorphisms (SNPs) were detected by PCR-RFLP method and the genotype of the above mentioned deletions was determined by touchdown PCR. Some bioinformatics tools were employed to evaluate the impact of SNPs on the gene function. SNP analysis revealed that there are significant associations between rs1048943 (AG vs. AA: OR = 2.46, 95%CI = 1.30–4.65, p = 0.006; GG + AG vs. AA: OR = 2.53, 95%CI = 1.36–4.69, p = 0.003; G vs. A: OR = 2.36, 95%CI = 1.33–4.17, p = 0.003) and rs4646903 (C vs. T: OR = 1.45, 95%CI = 1.02–2.06, p = 0.040) variations and increased risk of presbycusis. However, there was no significant association between rs1695 and presbycusis risk. Also, significant associations were observed between GSTM1 (OR = 4.28, 95%CI = 1.18–15.52, p = 0.027) and GSTT1 (OR = 1.64, 95%CI = 1.02–2.65, p = 0.041) deletions and elevated risk of presbycusis. Moreover, the combination analysis revealed a significant association between GSTM1+/GSTT1− genotype and presbycusis susceptibility (OR = 1.63, 95%CI = 1.00–2.67, p = 0.049). In silico analysis revealed that the rs1048943 SNP could influence significantly on the RNA structure of CYP1A1 (distance: 0.1454; p value: 0.1799). Based on our findings, the rs4646903,
