Photodynamic antimicrobial chemotherapy (PACT) is a novel alternative antimicrobial therapy that elicits a broad mechanism of action and therefore has a low probability of generating resistance. Such properties make PACT ideally suited for utilization in localized applications such as burn wounds. The aim of this study was to determine the antimicrobial activity of MB and temoporfin against both a S. aureus isolate and a P. aeruginosa isolate in light (640 nm) and dark conditions at a range of time points (0-20 min). A Staphylococcus aureus isolate and a Pseudomonas aeruginosa isolate were treated in vitro with methylene blue (MB) and temoporfin under different conditions following exposure to light at 640 nm and in no-light (dark) conditions. Bacterial cell viability [colony-forming units (c.f.u.) ml-1] was then calculated. Against P. aeruginosa , when MB was used as the photosensitizer, no phototoxic effect was observed in either light or dark conditions. After treatment with temoporfin, a reduction of less than one log (7.00×107 c.f.u. ml-1) was observed in the light after 20 min of exposure. However, temoporfin completely eradicated S. aureus in both light and dark conditions after 1 min (where a seven log reduction in c.f.u. ml-1 was observed). Methylene blue resulted in a loss of S. aureus viability, with a two log reduction in bacterial viability (c.f.u. ml-1) reported in both light and dark conditions after 20 min exposure time. Temoporfin demonstrated greater antimicrobial efficacy than MB against both the S. aureus and P. aeruginosa isolates tested. At 12.5 µM temoporfin resulted in complete eradication of S. aureus . In light of this study, further research into the validity of PACT, coupled with the photosensitizers (such as temoporfin), should be conducted in order to potentially develop alternative antimicrobial treatment regimes for burn wounds.