Objective: The surface of provisional restorations applied before conventional or implant- supported fixed restorations may cause bacterial or fungal biofilm formation. The aim of this study was to evaluate the antimicrobial activity of acrylic resins used in provisional restorations modified with chlorhexidine diacetate. Methods: 120 cylindrical, auto-polymerized resin samples modified with chlorhexidine diacetate were prepared at concentrations of 0 (control), 1, 3, 5 wt %. The antimicrobial activity was examined against Streptococcus mutans, Enterococcus faecalis, and Candida albicans using Crystal Violet quantification, MTT assay, and Scanning Electron Microscopy. Data were analyzed by ANOVA and paired sample t-tests (α=0.05). Results: The addition of chlorhexidine diacetate influenced the growth rate and metabolic activity of microorganisms. The antimicrobial effect against C. albicans and S. mutans statistically increased with the percentage of chlorhexidine diacetate. E. faecalis bacteria were less affected by chlorhexidine diacetate compared to other pathogens. Conclusion: It has been shown that the effectiveness of CHDA in inhibiting the proliferation of microorganisms correlated positively with increasing concentration levels. More research is needed to confirm the impact of different chlorhexidine concentrations on the mechanical properties, clinical efficacy, and antimicrobial properties of CDHA