Staphylococcus aureus and Clostridium perfringens are two major bacteria that infect open wounds and delay the healing process. The rapid and progressive deterioration of soft tissue during S. aureus and C. perfringens coinfections is due to analogous necrotic alpha toxins produced by the two organisms. The aim of this study was to determine the alpha toxins of S. aureus and C. perfringens by duplex PCR. The PCR assay employed two sets of primers: hlaf/r to amplify staphylococcal alpha toxin gene hla (274 bp) and cpaf/r to amplify clostridial alpha toxin gene cpa (398 bp) along with a competitive internal amplification control (608 bp), simultaneously. Optimization of the duplex PCR assay was achieved by a modified Taguchi method, an engineering optimization process, in a nine-tube combinatorial array. The detection level of the duplex PCR was found to be 10 pg of purified DNA or 103 CFU mL−1 of S. aureus and 100 pg of purified DNA or 104 CFU mL−1 of C. perfringens. Other bacteria routinely found in tissue infections were tested for cross-reactivity and the duplex PCR turned out to be highly specific. This duplex PCR assay provides a rapid, robust and reliable alternative to the existing conventional techniques in establishing the aetiology of S. aureus and C. perfringens in soft tissue infections.