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Abstract Aim: to assess the antimicrobial effect of diode laser in deep carious lesions after step wise excavation of the tooth using bacteriological assessment method. Methodology: The study was a randomized, split mouth design, clinical trial. Thirty permanent molar in fifteen participants with deep occlusal carious lesions were randomly assigned into two groups (15 teeth each) group 1: Ca2OH stepwise excavation, group 2: laser disinfection. After cavity preparation, dentin samples collected at baseline and after immediate disinfection with diode laser device and after six months follow up. Another dentin sample was collected from both Ca2 OH stepwise excavation and laser disinfection groups. These samples were subjected to microbiological evaluation, Streptococcus mutans on mitis salivarius (MS) agar, and Lactobacilli (LB) on Lactobacillus MRS agar. Results: The results showed that diode laser has a significant difference from Ca2OH stepwise excavation group as it has a significant reduction on Streptococcus mutans and Lactobacillus counts in the laser disinfection group. Conclusions: The antibacterial effect of diode laser in the deep occlusal cavities was significantly different from Ca2OH excavation group. Diode laser disinfection method demonstrated successful clinical measurement when accompanied by step wise technique |