Background: An increasing prevalence of Candida infections has emerged with the wide use of immune-suppressants and antibiotics. Current antifungal drugs exhibit low efficiency and high toxicity to the normal organs. Photodynamic inactivation (PDI) provides an alternative therapeutic strategy involving the use of photosensitizer (PS) and light irradiation. This study evaluated PDI effects against strains of C. albicans, C. parapsilosis, C. krusei and C. glabrata, using the PS of hematoporphyrin monomethyl ether (HMME), which is a second-generation PS clinically approved in China. Methods: Suspensions (~106 CFU/ml) were incubated with seven HMME concentrations (0.25~50 μM) for 30 min followed by 532-nm laser irradiation for 10 min at 40 mW/cm2. Viability of cells was assayed by CFU counting. Furthermore, fetal calf serum (10%) and singlet oxygen quencher sodium azide (100mM) were respectively added to the suspension of C. krusei to evaluate their roles in PDI process. Results: Among the four species, C. albicans was the most sensitive to PDI; 4 log10 killing was achieved at the concentration of 7.5 μM. C. glabrata was the most resistant; 3 log10 killing was not obtained even at PS concentration of 50 μM. PDI effects against C. krusei were inhibited by both serum and sodium azide. Conclusions: HMME-mediated PDI was able to effectively kill Candida in our experimental conditions, mainly through a Type Ⅱ photoprocess. However, the effects could be intensively reversed by the presence of serum. Thus, there might be a long way before HMME can be used in fighting against Candida in real infectious foci.