The growing resistance against antifungal agents has renewed the search for alternative treatment modalities, and antimicrobial photodynamic inactivation (PDI) is a potential candidate. The cationic porphyrin 5-phenyl-10,15,20-Tris(N-methyl-4-pyridyl)porphyrin chloride (TriP) is a photosensitizer that in combination with light can inactivate bacteria, fungi, and viruses. For future improvement of the efficacy of PDI of clinically relevant fungi such as Candida albicans, we sought to understand the working mechanism by following the response of C. albicans exposed to PDI using fluorescence confocal microscopy and freeze-fracture electron microscopy. The following events were observed under dark conditions: TriP binds to the cell envelope of C. albicans, and none or very little TriP enters the cell. Upon illumination the cell membrane is damaged and eventually becomes permeable for TriP. After lethal membrane damage, a massive influx of TriP into the cell occurs. Only the vacuole membrane is resistant to PDI-induced damage once TriP passes the plasma membrane. Increasing the incubation time of C. albicans with TriP prior to illumination did not increase the influx of TriP into the cell or the efficacy of PDI. After the replacement of 100% phosphate-buffered saline (PBS) by 10% PBS as the medium, C. albicans became permeable for TriP during dark incubation and the efficacy of PDI increased dramatically. In conclusion, C. albicans can be successfully inactivated by the cationic porphyrin TriP, and the cytoplasmic membrane is the target organelle. TriP influx occurred only after cell death.