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Purpose: To evaluate the antimicrobial action and elemental composition of chlorhexidine (CHX) diacetate in acrylic resins based on PMMA (polymethyl methacrylate) in situ. In addition, ex vivo evaluation of the CHX release mechanism was performed over a 14-day period., Methods: Three discs of PMMA incorporating CHX and three control discs were mounted on individual oral splints and exposed to the oral cavity of 32 participants for 24 hours. The antimicrobial activity was evaluated by the plate count method. In the second test, elemental analysis of the specimens (n = 10) was performed by X-ray fluorescence before and after use of the device. Chlorhexidine release over a 14-day period was evaluated ex vivo in saliva samples collected from five individuals through proton nuclear magnetic resonance spectroscopy ( ¹H NMR) (500 MHz)., Results: Bacterial adhesion, evaluated by the plate count method, did not differ between the experimental material and control. (P> 0.05) The presence of the CHX molecule was detected by X-ray fluorescence before and after insertion of discs containing CHX into the oral cavity of participants. With regard to release, CHX was detected in saliva samples for 14 days and highest during the first 24 hours. When partial least squares discriminant analysis (PLS-DA) was applied in ¹H NMR, we observed a greater difference between the test and control groups., Clinical Significance: The sustained release of CHX from PMMA suggests that such materials may be convenient for reducing the development of biofilm on the surface of the material for use in dentures and temporary restorative materials., Competing Interests: The authors declared no conflict of interest., (Copyright©American Journal of Dentistry.)