Objective: The objective of the present study was to evaluate the microbiological effectiveness of the glass ionomer cement added by 1% chlorhexidine diacetate. Methods: The antimicrobial activity was performed through the test of halo formation to inhibit Streptococcus mutans (S. mutans), Staphylococcus aureus (S. aureus) and Candida albicans (C. albicans) in the following groups: Group 1: glass ionomer cement (GIC), Group 2: GIC added by 1% chlorhexidine diacetate, Group 3: chlorhexidine digluconate solution at 0.12% and Group 4: chlorhexidine digluconate solution at 2%. The halo inhibition area was measured in square millimeter (mm²), at 24 and 48 hours after incubation (37o.C). Results: The chlorhexidine digluconate solution at 2% (group 4), used as a positive control had a significantly higher antimicrobial effect than the other groups (groups 1, 2 and 3) and against all the microorganisms tested (p<0.01, Kruskal-Wallis test). The glass ionomer cement added by 1.0% chorhexidine diacetate (group 2) showed a statistically higher antimicrobial effect against S. aureus and S. mutans species than groups 1 and 3 (p<0.01). Conclusion: It was concluded that the addition of 1% chlorhexidine diacetate enriched GIC functions demonstrated by the effectiveness in growth inhibition of Streptococcus mutans and Staphylococcus aureus.