Illegal artisanal mining, or ‘Galamsey’, in Ghana poses serious public health risks by contaminating water bodies with toxic heavy metals. This study assessed the sub-chronic toxicological effects of mining-polluted rivers in four Ghanaian regions using rats as an experimental model. A 90-day sub-chronic toxicity test was conducted using water samples from four regions associated with mining-polluted rivers: Bonsa, Birim, Nyam, and Subiri. Heavy metal analysis was conducted on the sampled water from these selected illegal mining-prone rivers. Rats were divided into six groups: two controls (distilled water and pristine river) and four treatment groups (contaminated water from the four rivers). Rats were given 2 mL of water from each water source for 90 days. Blood samples were analyzed for hormonal, hematological, and several biochemical markers. Fasting blood sugar, urine, and semen analyses were also performed. Rats were euthanized, and organs were harvested for histopathological analysis. Lead was significantly elevated in all mining-polluted rivers when compared to the controls. Exposed groups showed significant weight loss, elevated serum creatinine kinase (CK-Nac), and the presence of WBC, RBC, crystals, and casts during urinalysis. Haematological profiles indicated erythrocytosis and immune variations. Serum biochemical parameters, such as AST, ALP, and ALT, were significantly elevated (p < 0.05), and sperm counts were significantly reduced (p < 0.05) in the exposed groups. Histopathology revealed non-caseating granulomas, necrosis, and leucocytosis in the liver and lungs, as well as pronounced hemosiderosis in the spleen. Histopathology of renal and testicular organs revealed necrotic tissue and degenerative cellular changes.