Gentamicin is considered one of the most typical causes of testicular damage. Oxidative stress is a significant contributor to testicular tissue damage. Zamzam water (alkaline in nature) has an antioxidant effect. The purpose of this study was to assess the potential palliative effect of Zamzam water against gentamicin-induced testicular damage. Thirty Rats were separated into three groups, each with ten rats, as follows: The Control received only normal saline. The gentamicin group received 100 mg/kg/day of gentamicin intraperitoneally for six days from day 15 to the end of the experiment. The gentamicin +Zamzam Water group received a dose of gentamicin 100 mg/kg/day intraperitoneally with Zamzam water as their sole source of drinking from day one to day 21. Hormonal assay in serum, histological, immunohistochemical, and ultrastructural examination of testicular tissue with a molecular study were obtained. Pretreatment with Zamzam water significantly p < 0.001 increased serum levels of testosterone, FSH, and LH, as well as the percentage of sperm motility and progressive motility. It also upregulated SOD, CAT, GPx enzymatic activity, gene expression of Nrf2/HO-1, and immunoexpression of PCNA. While the percentage of dead sperm and abnormal sperm, immunoexpression of NFκB, Caspase 3, inflammatory cytokines TNFα, IL-1β, IL-6, and MDA levels significantly (p < 0.001) declined with histological improvement. It was concluded that Zamzam water as alkaline water possesses antioxidant, anti-inflammatory, and antiapoptotic effects against gentamicin-induced testicular toxicity in vivo.