The aim of this study was to determine the photodynamic antimicrobial effect of hypericin on clinically isolated Staphylococcus aureus and Escherichia coli cells. Bacterial cells (108 cells per mL) were incubated with hypericin (0–40 μm) for 30 min and followed by light irradiation of 600–800 nm at 5–30 J cm−2. Cell survival was determined by colony counting, cellular hypericin uptake examined by flow cytometer, and cell membrane damage examined by scanning electron microscopy and leakage assay. The effectiveness of hypericin-mediated photodynamic killing was strongly affected by cellular structure and photosensitizer uptake. The combination of hypericin and light irradiation could induce significant killing of Gram positive methicillin-sensitive and -resistant S. aureus cells (>6 log reduction), but was not effective on Gram negative E. coli cells (<0.2 log reduction). The difference was caused by different cell wall/membrane structures that directly affected cellular uptake of hypericin.