Hepatitis E virus (HEV) is the most common cause of endemic and epidemic acute hepatitis. A correlation between iNOS, eNOS polymorphisms, levels and severity of disease has been reported, and here, we examined the role of iNOS and eNOS gene polymorphisms and their levels in HEV-related acute viral hepatitis and acute liver failure. Hepatitis E virus-related cases of acute hepatitis (294 patients) and liver failure (82 patients) and age- and sex-matched healthy controls (331 subjects) were included in the study. PCR-RFLP was performed to identify the polymorphisms in the iNOS and eNOS genes. iNOS and eNOS levels were studied using ELISA assays and HEV viral load, genotype and combined effects of iNOS genotype, levels and parameters for disease severity were examined. The frequency of iNOS (CT + TT) and eNOS (GT + TT) genotypes was higher in subjects with liver failure compared with controls. iNOS and eNOS levels in patients with acute liver failure (55.51 ± 6.33 IU/mL, 60.2 ± 3.69) cases were significantly increased as compared to patients with acute viral hepatitis (17.8 ± 6.08 IU/mL, 23.7 ± 6.57) and controls (P < 0.05). A significant positive correlation was observed between the iNOS and eNOS levels in our study population when compared with the severity of disease parameters. Hence, the iNOS C150T polymorphism and the eNOS G894T polymorphism and high levels of iNOS and eNOS are associated with an increased risk of HEV-related acute hepatitis and liver failure. This study supports the possible role of nitric oxide synthase genes (iNOS and eNOS) in determining the severity of HEV infection.