The photodynamic activity of 5,10,15,20-tetra(4-N-methylpyridyl)porphyrin (TMPyP) was evaluated in vitro on Candida albicans cells under different experimental conditions. This tetracationic porphyrin binds rapidly to C. albicans cells, reaching a value of ∼1.7 nmol 10−6 cells when the cellular suspensions (106 CFU mL−1) were incubated with 5 μM sensitizer. The amount of cell-bound sensitizer is not appreciably changed when cultures are incubating for longer times (>15 min) but it diminishes with the number of washing steps. Photosensitized inactivation of C. albicans cellular suspensions increases with both sensitizer concentration and irradiation time, causing a ∼5 log decrease of cell survival when the cultures are treated with 5 μM TMPyP and irradiated for 30 min. However, the photocytotoxicity decreases after one washing step, with the decrease in cell-bound sensitizer. The growth of C. albicans cells was arrested when the cultures were exposed to 5 μM TMPyP and visible light. On agar surfaces, the phototoxic effect of this sensitizer, which caused an inactivation of C. albicans cells, remained high. No growth was observed in areas containing TMPyP and irradiated. Moreover, in small C. albicans colonies, C. albicans cells were completely inactivated. These studies indicate that TMPyP is an effective sensitizer for photodynamic inactivation of yeasts in both liquid suspensions or localized on a surface.