Streptococcus mutans is one of the most significant contributors to tooth decay. Recently, blue light was reported to have a bactericidal effect. This study aims to evaluate the bactericidal effect of an LED toothbrush and determine the effect of duration of LED exposure on Streptococcus mutans biofilm in vitro. The LED light source is from an LED toothbrush (WHITENGO^TM, UK). S. mutans biofilms were assigned to 5 groups with 6 samples each, depending on the duration of LED exposure, which are 15 seconds, 30 seconds, 60 seconds, 120 seconds, and the control with no LED exposure. Kruskal-Wallis test was performed to compare the percentage of viability among groups. The percentage of bacterial viability in the 120-second group was significantly lower than those of other groups. The LED toothbrush effectively reduced S. mutans viability when the biofilm was exposed to the light for at least 120 seconds. This information may help consider the use of the LED toothbrush for oral care.