Present study was conducted with the aim of evaluating antimicrobial efficacy of two activation irrigating techniques namely; (Nd:YAG) laser and EndoVac with 5.25% sodium hypochlorite, against biofilm of two types of microbial strain, Candida albicans (ATCC 14053) and Enterococcus faecalis (ATCC 19433). In total, the root canals of 40 extracted single-rooted human mandibular premolar teeth were prepared using the ProTaper Next system rotary instrument up to X4 (# 40). Samples were divided randomly in 8 groups (n=5 each). Negative group teeth were checked for their sterility. Then, the remaining teeth were inoculated with Enterococcus faecalis and Candida albicans and incubated at 37°C for 48 hours to isolate the bacteria. Then the new strains were cultured and adjusted at 10 6-10 8 CFU/mL according to 0.5 MacFarland standard using a spectrophotometer at an optical density (OD 595). After three weeks of incubation in respective media for each microbe, the formation of biofilms was evaluated and the OD 595 of both E. faecalis and Candida albicans were measured by spectrophotometer as a positive control of the test. Then, the remaining samples were irrigated with the designed irrigation technique for each group. Then the turbidity of the growth was determined by Spectrophotometer at 595 nm of each group. Significant difference was found among all the groups in comparison to the control group. Both irrigation technique showed significant reduction in bacterial counts post-treatment, but the greatest reduction was in the laser treatment of the E. faecalis infected group.