BACKGROUND Substantivity is one of the desirable characteristics of root canal irrigants. Among conventional endodontic irrigants, only chlorhexidine (CHX) is known to exert significant substantivity on root canal dentin. This study explored the substantivity activity of a polymeric biguanide-poly hexamethylene biguanide (PHMB). OBJECTIVE The aim of this study was to determine the substantivity of a polymeric biguanide - 0.2% PHMB and compare it with that of 2% CHX. METHODS To evaluate and compare the substantivity of 0.2% PHMB and 2% CHX on root canal dentin, dentin disks were prepared and substantivity after 1 h, 24 h, 7 days and 21 days was measured using spectrophotometry. METHODS The data so obtained were analyzed, and the intergroup comparison was made using unpaired t-test. RESULTS The results of this study indicated that 0.2%. PHMB exerts significantly greater substantivity than 2% CHX on human dentin. CONCLUSIONS Within the limitations, this study supports the use of 0.2% polyhexanide as an endodontic irrigant based on its property of substantivity.