The objective of this study was to test the effectiveness of 0.2% polyhexamethylen biguanide in the elimination of Enterococcus faecalis, Pseudomonas aeruginosa and Candida albicans and compare it with effectiveness of 2.5% sodium hypochlorite. Crown and apical portion of forty- eight single-rooted human teeth was removed and the root canals instrumented with Hedström files up to size 40. Sixteen specimens were inoculated with an overnight broth culture of E.faecalis (ATCC 51299), P.aeruginosa (ATCC27853) and C.albicans (clinical isolate). After 48 h incubation and irrigation with saline, dentine samples were collected from root canal walls with Hedström files size 50 before and after irrigation with 0.2% PHMB and 2.5% NaOCl followed by irrigation with neutralizer and saline. Samples were put into vials containing 2 mL of sterile saline and tenfold dilution, and plated on blood agar after 30 s vortexing. Colony forming units (CFU) were counted after 48 h incubation at 37ºC. For statistical analysis Kruskal-Wallis test (p<0.05) was used. Results: After treatment with PHMB P.aeruginosa was eradicated from all samples, E.faecalis was also reduced and C.albicans was not grown in seven out of eleven samples. NaOCl was less effective than PHMB on E.faecalis (p=0.630) and P.aeruginosa (p=0.138), especially in case of C.albicans (p=0.01). 0.2% PHMB effectively reduced the number of E.faecalis and P.aeruginosa as well as 2.5% NaOCl). On C.albicans 0.2% PHMB showed better antimicrobial effect than 2.5% NaOCl. Further studies will be necessary to determine PHMB's effectiveness on mature biofilm and its biocompatibility.