AIMS To compare antibacterial effect and physical properties of chitosan (CH) modified glass ionomer cement (GIC) (10% v/v), chlorhexidine-cetrimide (CHX-CT) modified GIC (2.5/2.5% w/w) and conventional GIC. MATERIALS AND METHODS A total of fifty healthy children of age 7-12 years were selected and randomly assigned to class A and B for in vivo analysis. Slabs of CH modified GIC (Group II) along with slabs of conventional GIC (Group I, control) were cemented on buccal surfaces of maxillary molars (split-mouth technique) for class A children. Similarly, slabs of CHX-CT modified GIC (Group III) were cemented against control (Group I, control) in class B children. Slabs were assessed after 48 h for microbial load of Streptococcus mutans and Lactobacillus (LB) on mitis salivarius-bacitracin and Man Rogosa Sharpe agar media, respectively. Agar diffusion test was done to access the antibacterial effect of each group against Streptococcus muatns and LB. Slabs and cylinders of GICs were made for in vitro evaluation of compressive and flexure strength in each group. RESULTS Comparison was done by nonparametric Kruskal-Wallis analysis followed by Dunn's multiple comparison test. Categorical groups were compared by Chi-square test. The increase in antibacterial activity (Group II > III > I) (P < 0.001) and marked increase in compressive and flexure strength (Group II > I > III) were observed. CONCLUSIONS In the view of findings, it is concluded that CH modified GIC would be effective in inhibiting the bacteria associated with dental caries along with improved physical properties when compared with CHX-CT modified GIC and conventional GIC.