Aim: To evaluate an inter-generic recombinant alpha domain fusion protein for simultaneous detection and neutralization of Clostridium perfringens and Staphylococcus aureus alpha toxins. Methods and Results: Truncated portions of clostridial and staphylococcal alpha haemolysin genes were PCR amplified and linked to each other through a hydrophilic flexible Glycine linker sequence using overlap-extension PCR to form a chimeric gene aCS. The recombinant aCS fusion protein was expressed and characterized for its toxicity, cell binding capacity and haemolysis inhibition properties. The fusion protein was nontoxic and effectively retarded staphylococcal alpha haemolysis, probably by competitively interacting with putative staphylococcal alpha haemolysin receptors on erythrocytes. Murine hyperimmune polysera raised against r-aCS specifically detected 42-kDa and 33-kDa proteins when culture supernatants of Cl.similar to perfringens (clostridial alpha toxin) and Staph.similar to aureus (staphylococcal alpha toxin), respectively, were analysed in Western blot. The polyclonal antisera effectively diminished the haemolytic action of both the wild-type toxins in vitro. Conclusions: The r-aCS fusion protein was nontoxic competitive inhibitor of staphylococcal alpha haemolysin. The protein elicited specific immune response against Cl.similar to perfringens and Staph.similar to aureus alpha toxins. The antisera also neutralized the toxicities of both the native wild-type toxins in vitro. Significance of the Study: The bivalent recombinant aCS protein could be a novel intervention in the field of diagnostics and therapeutics against Cl.similar to perfringens and Staph.similar to aureus infections, particularly, in case of co-infections like gangrenous ischaemia, gangrenous mastitis, etc.