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Introduction: Glass-ionomer (GIC) cement was introduced in 1972 as a "new filling material of dentistry". It is bioactive and plays an important role in caries prevention due to its ability to release fluoride into the oral environment and remineralization of dental hard tissues. However, its properties such as moisture sensitivity, wear resistance, and bond strength are not sufficient to inflict the antimicrobial environment. This in vitro study aimed to evaluate the antibacterial property of four different GIC cements against S. mutans and L. acidophilus., Methodology: This study was conducted on 120 disk-shaped samples (30 for antibacterial activity), which were placed in Petri dishes holding Müeller Hinton agar. Bacterial strains were overhauled in the brain heart infusion culture medium, and by utilizing disposable straps on blood agar medium, 100 ml of the strain inoculum was plated out. Through the diffusion method on the solid medium, the antibacterial activity of GIC was determined., Results: The antibacterial activity was the highest for Riva silver and chemifill rock for 24 and 72 hours, respectively. For 48 hours, Equia forte and chemifill rock had the highest antibacterial activity, and there was a significant difference between the groups., Conclusion: Ketac™ molar easymix inhibited the growth of S. mutans and L. acidophilus but had the lowest antibacterial effect compared to other GICs., Competing Interests: There are no conflicts of interest., (Copyright: © 2024 Journal of Pharmacy and Bioallied Sciences.)