Purpose The objective of this study was to evaluate the antibacterial activity (ABA), Vickers microhardness numbers (VHN) and cumulative fluoride-releasing (CFR) patterns of conventional glass ionomer cement (GIC) containing AB agents. Methods Chlorhexidine (CHX), Cetrimide (CT) and Cetylpyridinum Chloride (CPC) were added to the powder and Benzalkonium Chloride (BC) was added to liquid of GIC in concentrations of 1% and served as the experimental group (EG). Antibacterial-free GIC was used a control group (CG). Results Compared to the CG, a statistically significantly higher level of ABA was detected at the 1st and 7th day against Streptococcus mutans (SM) and on all days against Lactobacillus casei (LC). The CG had statistically significantly high microhardness values in all time periods compared to the EG. With regard to fluoride ion release, there was no statistical difference between CG and EG at all times. A statistically significant increase was observed in both CG and EG during the 1st day to the 30th day. Conclusion The results of this in vitro investigation demonstrated that AB agents added to the GIC can exhibit AB effects against SM and LC without seriously damaging the physical and chemical properties of the material.