When more than one species of bacteria infects a wound in the body, they form a polymicrobial biofilm. Studies have found that multiple strains of bacteria grow synergistically with one another, amplifying not only their toxicity but also their resistance to treatment. Polymicrobial chronic wound infections are fatal for many patients due to their invasiveness and resistance to antibiotics. To provide a better treatment plan for chronic wound infections, an in vitro model must first be established for extensive research. Staphylococcus aureus and Pseudomonas aeruginosa have been identified as common bacterial inhabitants of polymicrobial biofilms. Based on this data, it is ideal to establish an in vitro model consisting of both S. aureus and P. aeruginosa. Issues arise when growing the two species in vitro because in a one to one ratio, P. aeruginosa dominates S. aureus, resulting in a single species biofilm or no biofilm at all. The aim of this study is to find the optimal conditions between S. aureus and P. aeruginosa to grow an in vitro polymicrobial biofilm representative of an in vivo polymocrobial biofilm. This was achieved by inoculating them at different ratios and determining the optimal condition that results in growth of both bacteria.