Purpose The purpose of the study was to explore the possible deleterious effects of Methotrexate (MTX) treatment on the urogenital tissues and the potential protective effects of Astaxanthin (AXA). Methods Twenty-four female Wistar Albino rats (12 months old) were divided into 3 groups as follows: Group I (Control group): rats received a single dose of 0.1 ml saline by gavage and intraperitoneal injection. Group II (MTX group): rats received a single dose of 20 mg/kg MTX, i.p, on the 2nd day. Group III (MTX + AXA group): rats received 100 mg/kg AXA orally for 7 days in addition to a single dose of MTX. The levels of total oxidant status (TOS), total antioxidant status (TAS), oxidative stress index (OSI), and histopathological and immunohistochemical markers (Caspase-3, iNOS, CRP, G-CSF) were evaluated in urogenital tissues. Results In ovarian tissues, a statistically significant increase in TOS levels (p = 0.001) and OSI index (p = 0.028) were observed in Group II compared to Group I. TAS level was significantly higher in Group III compared to Group II and I (p = 0.009 and 0.002, respectively). However, a significant decrease in OSI level was observed in Group III compared to Group II (p = 0.035). In fallopian tube tissues, TAS level was significantly decreased in Group II compared to Group I (p = 0.047). Histopathologically, marked hyperemia was observed in MTX group. AXA treatment ameliorated all the pathological findings. Immunohistochemically, all the studied markers were considerably increased in Group II, however, they were decreased by AXA. Conclusion These findings revealed that MTX treatment caused oxidative stress, apoptosis, and inflammation in the urogenital tissue. We found that AXA significantly ameliorated the damage caused by MTX in the urogenital tissue. The results of the study have indicated that AXA may be a promising nutritional support substance against the damage caused by chemotherapeutic and cytotoxic agents, such as MTX, to the urogenital tissue.