The minimum inhibitory concentration (MIC) of chlorhexidine for Staphylococcus aureus, Bacillus subtilus, Escherichia Coli and Candida albicans was studied. Chlorhexidine polymeric films composed of ethylcellulose (EC) and hydroxypropyl methyl- cellulose (HPMC) in a ratio of 8:2 were prepared. The inhibition zone diameters of chlorhexidine polymeric film were compared with that of chlorhexidine gauze dressing. Also, the inhibition zone sizes of different concentrations of chlorhexidine (0.5, 1 and 5%) in films plasticized with 20% propylene glycol, in presence of 10% Tween 80 as enhancer, or plasticized with 20% polyethylene glycol 400 were evaluated. Results indicated that the MIC of chlorhexidine was ranged from 1 to 1.8 ug/ml. Also the inhibition zone diameters of the selected polymeric film was higher than that of gauze dressing. There were correlation between drug concentrations and inhibition zone sizes. Also polymeric film containing 20% propylene glycol and 10% Tween 80 showed relatively higher response than polymeric film containing 20% ployethylene glycol 400. It can be concluded that the tested microorganisms were susceptible to chlorhexidine and the use of polymeric films as drug delivery system enhanced chlorhexidine in-vitro antimicrobial activity. Accordingly, it was recommended to use chlorhexidine polymeric films for topical antisepsis.