Aim: To evaluate and compare the antibacterial efficacy and horizontal depth of penetration of various irrigants into the dentinal tubules when used alone and when combined with lasers. Materials and methods: An experimental study was done on 42 single-rooted teeth. Access cavity preparation was done and the canals were enlarged up to a ProTaper file size F2 of length 25 mm. They were inoculated with 0.1 mL of Enterococcus faecalis and the samples were randomly assigned into six different groups of seven teeth each. The following irrigation systems were used individually and in combinations—normal saline, sodium hypochlorite, chlorhexidine gluconate, diode laser and erbium, chromium:ytrrium scandium gallium garnet laser (Er,Cr:YSGG laser). The colony-forming units (CFU) of bacteria before and after disinfection and the penetration depth of different groups were determined. Statistical analysis was done by an ANOVA test. Results: The highest number of CFU of bacteria was shown by the group where saline was used and it also showed the least penetration depth compared to that of the Er,Cr:YSGG laser group. Conclusion: Er,Cr:YSGG laser when used along with sodium hypochlorite and chlorhexidine gluconate showed the highest reduction in the CFU of bacteria and the greatest penetration depth when observed under a stereomicroscope. Clinical significance: Laser-assisted irrigation regimes have a high antibacterial efficacy and more penetration depth into the dentinal tubules.