Aims: The goal of this investigation was to develop an in vitro, polymicrobial, wound biofilm capable of supporting the growth of bacteria with variable oxygen requirements. Methods and Results: The strict anaerobe Clostridium perfringens was isolated by cultivating wound homogenates using the drip-flow reactor (DFR), and a three-species biofilm model was established using methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa and Cl. perfringens in the colony-drip-flow reactor model. Plate counts revealed that MRSA, Ps. aeruginosa and Cl. perfringens grew to 7.39 +/- 0.45, 10.22 +/- 0.22 and 7.13 +/- 0.77 log CFU per membrane, respectively. The three-species model was employed to evaluate the efficacy of two antimicrobial dressings, Curity (TM) AMD and Acticoat (TM), compared to sterile gauze controls. Microbial growth on Curity (TM) AMD and gauze was not significantly different, for any species, whereas Acticoat (TM) was found to significantly reduce growth for all three species. Conclusions: Using the colony-DFR, a three-species biofilm was successfully grown, and the biofilms displayed a unique structure consisting of distinct layers that appeared to be inhabited exclusively or predominantly by a single species. Significance and Impact of the Study: The primary accomplishment of this study was the isolation and growth of an obligate anaerobe in an in vitro model without establishing an artificially anaerobic environment.