Objective: To evaluate the in vivo antimicrobial activity of different forms of application of chlorhexidine digluconate (CHX). Method and Materials: A group of 10 volunteers performed mouth rinsings with a 0.2% and 0.12% solution of CHX and applied CHX in the form of a 0.2% gel, a 0.2% and a 0.12% spray and a swab impregnated with a 0.2% solution. Samples of saliva were taken at baseline; 30 seconds; and 1, 3, 5, and 7 hours after each application. An epifluorescence microscopy technique (BacLight Live/Dead stain) was used to evaluate bacterial vitality. Results: Bacterial vitality under basal conditions was 92%. At 30 seconds after CHX application, the lowest levels of viable bacteria were detected with the 0.2% and 0.12% solutions (1% and 8%, respectively). After 3 hours, the percentage of live bacteria achieved with the gel and sprays was similar to the basal figures (80% to 91%). At 7 hours, bacterial vitality recovered basal percentages, except with the 0.2% solution (77%). Conclusion: The 0.2% solution of CHX had the greatest sustained antimicrobial effect on the salivary flora, confirming the influence of the concentration and form of application on CHX substantivity. (Quintessence Int 2009;40:141–144) Key words: chlorhexidine, oral flora, saliva, substantivity