Aim: The present study was conducted to evaluate and compare antimicrobial effect of 5.25% sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX) and N-Acetylcysteine (NAC) irrigating solutions against Enterococcus faecalis (E. faecalis) and Streptococcus mutans (S. mutans). Materials and methods: The present study was conducted on 40 freshly extracted noncarious permanent mandibular incisors teeth of both genders (males-12, females-14). In all teeth, root canal preparation was done up to size 40 K-file. Roots were sterilized and microbial suspension of mixed culture of the tested microorganisms was inoculated into canals and incubated for 48 h. Teeth were divided into four groups, group I (5.25% sodium hypochlorite), group II (2% chlorhexidine), group III (200 mg/mL N-Acetylcysteine NAC) and group IV (sterile distilled water). The antimicrobial effect in each group was compared. Results: Statistical evaluation was completed using statistical software Statistical Package for the Social Sciences (SPSS). Planktonic S. mutans bacterial count was lowest in group III followed by group I, group II and group IV. E. faecalis count was 6.14 ± 0.12 in group I, 5.76 ± 0.44 in group II, 3.88 ± 0.08 in group III and 11.98 ± 1.04 in group IV. The difference was significant (p < 0.05). The proportion of dead cell found to be 0.04± 0.01, 0.72 ± 0.06, 0.01 ± 0.06 and 1.02 ± 0.11 in groups I, II, III and IV respectively. The difference was significant (p < 0.05). Conclusion: NAC proved to be effective against E. faecalis and S. mutans. This solution can be considered alternative in root canal infections in addition with CHX and NaOCl. Clinical significance: Effectiveness of three different irrigating solutions was compared and NAC found to be more efficient in decreasing bacterial count. Hence, NAC can be precisely used in irrigating root canals to achieve optimal clinical outcomes particularly regarding reoccurrences of infections. Furthermore, NAC could be proved as a promising innovation in future endodontic methodologies.