In atraumatic restorative treatment (ART), caries removal with hand excavation 1 instruments is not as efficient as that with rotary burs in eliminating bacteria under the glass ionomer cements (GICs). Thus, different antibacterial agents have been used in recent studies to enhance the antibacterial properties of the GICs, without jeopardizing their basic physical properties. Objective: The objective of this study was to evaluate the effect of antibacterial agents on the surface hardness of a conventional GIC (Fuji IX) using Vickers microhardness [Vickers hardness number (VHN)] test. Material and Methods: Cetrimide (CT), cetylpyridinium chloride (CPC) and chlorhexidine (CHX) were added to the powder and benzalkonium chloride (BC) was added to the liquid of Fuji IX in concentrations of 1% and 2%, and served as the experimental groups. A control group containing no additive was also prepared. After the completion of setting reaction, VHN measurements were recorded at 1, 7, 15, 30, 60, and 90 days after storage in 37 degrees C distilled water. A one-way ANOVA was performed followed by a Dunnett t test and Tamhane T2 tests and also repeated measurements ANOVA was used for multiple comparisons in 95% confidence interval. Results: VHN results showed significant differences between the control and the experimental groups at all time periods (p<0.05 for all). Significant differences were observed between all study periods for individual groups (p<0.05). After 7 days, VHNs were decreased in all experimental groups while they continued to increase in the control group. BC and CHX groups demonstrated the least whereas CT and CPC groups exhibited most adverse effect on the hardness of set cements. Conclusions: Despite the decreased microhardness values in all experimental groups compared to the controls after 7 up to 90 days, incorporating certain antibacterial agents into Fuji IX GIC showed tolerable microhardness alterations within the limitations of this in vitro study.