Cyclophosphamide (CP) is a commonly used antineoplastic and immunosuppressive drug that has serious adverse effects, such as ovarian damage and infertility. The aim of this study was to find out how hesperidin (HSP), a naturally occurring flavonoid with well-established health benefits, can prevent ovarian damage brought on by CP Thirty-two adult female albino rats were divided into four groups: control, HSP-treated, CP-treated, and CP+HSP-treated. CP administration resulted in significant hormonal imbalances, including reduced level of prolactin, estrogen, FSH, and LH and decreased body and ovarian weight. Oxidative stress markers were elevated with increased ovarian malondialdehyde (MDA) and reduced antioxidant activity. Molecular analysis showed downregulation of hypothalamic (KISS-1, KISS-1r, and GnRH), hypophyseal (GnRHr) genes, and key ovarian steroidogenic enzymes. CP also upregulated ovarian P21 and NF-κB, increased immunological expression of P53 and iNOS, and caused significant histopathological and immunohistochemical changes in ovarian tissues. HSP co-administration has alleviated many of these adverse effects, improved hormonal balance, and reduced oxidative stress. This restored gene expression, and preserved ovarian structure. These results highlight the potential of HSP as a protective agent against CP-induced ovarian damage and infertility, offering promising implications for female fertility preservation during chemotherapy.