Snakebite envenoming is a major yet neglected tropical disease in sub-Saharan Africa, where antivenom efficacy is critically limited by intraspecific venom variation shaped by local ecological pressures. Nigeria’s sharply contrasting Sudan Savanna (North) and Lowland Rainforest (South) provide an ideal natural system to investigate this variation, yet a comparative analysis of its medically important snakes has been lacking. We conducted an integrated proteomic and functional characterization of venoms from the puff adder (Bitis arietans) and black-necked spitting cobra (Naja nigricollis) collected in Kaduna (North) and Ibadan (South). Using high-resolution LC-MS/MS, SDS-PAGE, and biochemical assays (phospholipase A₂, protease, fibrinogenolytic, hemolytic, and coagulation activities), we mapped region-specific venom compositions and characterized their functional activities. Bitis arietans displayed region-associated divergence: southern venom was enriched in serine proteases, whereas northern venom was dominated by lectins and distinct snake venom metalloproteinase isoforms. Naja nigricollis showed a conserved phospholipase A₂/three-finger toxins backbone, yet southern venoms exhibited elevated snake venom metalloproteinase III and L-amino acid oxidase. These molecular differences manifested functionally, with southern B. arietans venom showing higher protease activity than northern B. arietans, whereas southern and northern N. nigricollis venom exhibited similar protease activity but enhanced phospholipase A₂ activity in southern N. nigricollis. This work provides the first integrated proteomic and functional comparison of venoms from northern and southern Nigerian venom sample of B. arietans and N. nigricollis. While based on a limited number of individuals, the observed differences should be considered preliminary and indicative of potential regional trends rather than population-level characteristics.