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 micro‐organisms 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•), 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.