The aim of the present study was to analyse the sustain release of Chlorhexidine with Chitosan and to investigate the antimicrobial activity of 2% Chlorhexidine gel, 2% Chitosan gel and their combination against Candida albicans and Enterococcus faecalis. Sustain release of Chlorhexidine with Chitosan was determined using UV spectrophotometer. Then the inoculae of these organisms were used to make the lawn culture on sabouraud's dextrose agar and blood agar plates. Wells were prepared in these lawn cultures and filled with 2% Chlorhexidine gel, 2% Chitosan gel and their combination. The agar plates were incubated overnight at 37°C and the zone of inhibition was examined after 48 h. Release of Chlorhexidine with Chitosan was better than plain Chlorhexidine release. Combination of Chlorhexidine and Chitosan showed maximum inhibitory zone for C. albicans (25.2 mm) and E. faecalis (26.0 mm). Plain Chlorhexidine gel showed intermediate inhibitory zone for C. albicans (20.6 mm) and E. faecalis (21.4 mm) and plain Chitosan gel showed minimum inhibitory zone for C. albicans (16.6 mm) and E. faecalis (11.0 mm). Carbopol which served as control did not have any antimicrobial effect. The present study suggests that 2% Chlorhexidine gel in combination with 2% Chitosan gel has the highest antimicrobial effect against C. albicans and E. faecalis compared with 2% Chlorhexidine gel or 2% Chitosan gel alone. © 2008 The Authors. Journal compilation © 2008 Australian Society of Endodontology.