AIM: The study was done to assess the antimicrobial effectiveness of Chitosan Nanoparticles (CNPs), Chlorhexidine (CHX), and their combination (CHX/CNPs) versus that of Sodium hypochlorite (NaOCl) in patients with mandibular necrotic premolars and to evaluate their effects on post-operative pain after single-visit endodontic treatment. MATERIALS AND METHODS: Sixty patients with necrotic mandibular premolars were divided randomly to four groups (n = 15) according to the used irrigating solution. Instrumentation was done using rotary ProTaper files. During instrumentation, irrigation was done using 2.5% NaOCl; afterward, canals were flushed with sterile saline. A final flush with the study irrigants was done as follows: 3% CNPs for Group A, 2% CHX for Group B, CHX/CNPs for Group C, and 5.25% NaOCl for Group D. Samples were collected from root canals before and after canal preparation then cultured to assess the number of colony-forming units/ml. All patients were instructed to record their pre- and post-operative pain levels on a numerical rating scale. RESULTS: CNPs and CHX/CNPs were significantly more effective than either CHX or NaOCl; however, there was no significant difference between them against anaerobic bacteria. All tested irrigants were similarly effective against aerobic bacteria. CNPs and CHX/CNPs were associated with significantly lower post-operative pain levels in the first 24 h after treatment. CONCLUSIONS: CNPs and its combination with CHX are significantly more effective than both CHX and NaOCl against anaerobic bacteria isolated from necrotic mandibular premolars. Post-operative pain intensity was significantly lower with CNPs and CNPs/CHX combination than with either NaOCl or CHX.