Aims: The major objective of the study was to evaluate the enhanced germicidal effects of low-frequency pulsed ultraviolet A (UVA)-light-emitting diode (LED) on biofilms. Methods and Results: The germicidal effects of UVA-LED irradiation (365 nm, 0.28 mW cm(-2), in pulsed or continuous mode) on Candida albicans or Escherichia coli biofilms were evaluated by determining colony-forming units. The morphological change of microbial cells in biofilms was observed using scanning electron microscopy. After 5-min irradiation, over 90% of viable microorganisms in biofilms had been killed, and pulsed irradiation (1-1000 Hz) had significantly greater germicidal ability than continuous irradiation. Pulsed irradiation (100 Hz, 60 min) almost completely killed micro-organisms in biofilm (>99.9%), and 20-min irradiation greatly damaged both microbial species. Interestingly, few hyphae were found in irradiated Candida biofilms. Moreover, mannitol treatment, a scavenger of hydroxyl radicals (OH center dot), significantly protected viable micro-organisms in biofilms from UVA-LED irradiation. Conclusions: The study demonstrated that pulsed UVA-LED irradiation has a strong germicidal effect (maximum at 100 Hz, over 5-min irradiation) and causes the disappearance of hyphal forms of Candida. Significance and Impact of the Study: This study can assist in developing a low-frequency pulsed UVA-LED system to be applied to pathogenic biofilms for disinfection.