This research aimed to determine whether irradiation of 405 nm diode laser with chlorophyll as photosensitizer could degrade the extracellular polymeric substance (EPS) of Enterococcus faecalis (E. faecalis) biofilm. The material for this study needs 25 biofilm formed by E. faecalis was divided equally into five groups. The control negative group (C-) consisted of E. faecalis biofilm, the control positive group (C+) consisted of E. faecalis biofilm and chlorophyll photosensitizers, and the other three treatment group (T1, T2, T3) consisted of E. faecalis biofilm and chlorophyll photosensitizers. Each treatment groups were irradiated for 90 second (s) for T1 group, 105 s for T2 group, and 120 s for T3 group with 405nm diode laser. The degradation EPS of E. faecalis’ biofilm was determined using Confocal Laser Scanning Microscope (CLSM). Irradiation duration affected the degradation EPS of E. faecalis’ biofilm. Chlorophyll with 120 s laser irradiation showed significant degradation EPS of E. faecalis’ biofilm compared to other groups (p < 0.05). Irradiation of diode laser 405nm with chlorophyll photosensitizer 120 s could degrade EPS of E. faecalis biofilm up to 97.51%.