The purpose of this study was to evaluate the effect of diode laser irradiation on Enterococcus faecalis (E. faecalis) and its lipoteichoic acid (LTA). Ninety-six freshly extracted single-rooted teeth were divided into six groups, n = 8 per group. Groups 1, 2, 3, and 4 as laser group (810nm PILOT™ Diode Laser, 400μm fiber diameter, continuous mode, 30s time) with powers at 1.0W, 1.5W, 2.0W, and 2.5W respectively. Group 5 or positive control group (3ml of 1% sodium hypochlorite (NaOCl) irrigation) and group 6 or negative control group (3ml of normal saline (0.9% NaCl) irrigation). Root canal samples were collected before and after receiving laser irradiation and irrigation solution. Cultivable bacteria were determined by counting the colony (CFU/ml). Evaluation of temperature on the external root surface of teeth was done with K type thermocouple using laser at different powers. Enzyme-linked immunosorbant assay (ELISA) was performed to measure the LTA levels and the correlations between E. faecalis count, LTA levels, and rise in temperature were observed using Pearson's correlation test. E. faecalis LTA was subjected to laser irradiation and its structural damage was examined by thin layer chromatography (TLC). Compared with the control groups, all laser groups showed a decreased colony counts and decreased LTA levels with statistically significant difference (p˂0.05). The bactericidal effect and LTA reduction of laser was better at 2.5W power. Laser at 2.5W power had temperature rise of more than 7°C which is beyond the safe thermal threshold level. No statistically significant correlation was found between E. faecalis count, levels of LTA, and rise in external root surface temperature (p˃0.05). TLC results showed a structural damage in the glycolipid moiety of E. faecalis LTA. Diode laser can effectively reduce the E. faecalis count and its LTA levels.