Bungarus fasciatus is one of the medically important elapid snakes of South and South-eastern Asia and is responsible for several snakebite incidents some of which were fatal. In this study, the venom compositional variation of Bungarus fasciatus from three different geographical locations in eastern and north-east India: two adjacent villages of Hooghly (West Bengal), Aizawl (Mizoram) and Guwahati (Assam) are reported. In vitro biochemical assays exhibit variation in phospholipase A2 activity, fibrinogenolytic activity, caseinolytic activity and anti-coagulation activity. The immunoreactivity of three Indian polyvalent antivenoms against the venoms revealed incomplete recognition. Bharat Serums antivenom demonstrated that at a venom–antivenom ratio of 1:16, the antivenom exhibited different immunocapturing abilities for all the venom samples. The percentage of non-retained fractions was highest for Guwahati (60.00%) and lowest for Hooghly 1 (18.91%). The study demonstrates intra-population (or inter-individual) variation of B. fasciatus venom from two nearby locations of Hooghly (West Bengal), intra-specific variation of B. fasciatus from three geographical locations and also inter-specific venom variation with B. caeruleus from Tamil Nadu. Thus, the venom variation leads to partial immune cross-reactivity by Indian polyvalent antivenoms. Inclusion of non-recognized venom proteins in the immunization mixture during antivenom production would help to improve the efficacy of the antivenom. Further study of the neutralizing ability of Indian polyvalent antivenoms against medically important snakes from different geographical regions would help to understand the effectiveness of the antivenoms and would invariably assist in the designing and development of safe and effective antivenoms.