Introduction: The atraumatic restorative treatment (ART)considered a trusted and effective approach to the control of carious lesions.Since the ART approach using glass ionomer (GICs) the use of antimicrobial agents(chlorhexidine) in combination with restorative materials are being developed for reducing the frequency and severity of secondary caries. hence, it is preferable to keep an optimum chlorhexidine concentration to maintain its antimicrobial effect without affecting GICs’ mechanical performance. Aim of the study: This study was planned to evaluate the antibacterial activity and mechanical properties of the glass ionomer containing different concentrations of chlorhexidine. Material and methods: In vivo study: Sixty children aged 7-9 years old were included in this studywith at least onecavitated dentin carious lesion in occlusal surfacesin primary teeth.these children were randomly divided into three equal treatment groups. Group I: atraumatic restorative treatment approach using a conventional glass ionomer cement. Group II: atraumatic restorative treatment approach using glass ionomer containing1% chlorhexidine. Group III: atraumatic restorative treatment approach using glass ionomer containing 2% chlorhexidine. A sample was taken from the affected dentine using a sterile sharp small-size hand excavator at baseline and after 7 days. All samples were inoculated for the selective isolation of mutans streptococci, and lactobacilli. In vitro study: Fifteen cylindrical specimens for each group were prepared and tested for their compressive strength, diametral compressive strength and microhardness. Results: The percentage reduction in mean and standard deviation (SD) of mutans streptococci and lactobacilli between treatment groups revealed that there were statistically significant differences between three groups (P= 0.01, P= 0.02).Tukey post hoc test revealed that the main difference was between glass ionomer containing chlorohexidine and conventional glass ionomer while there was no statistically significant difference between glass ionomer containing 1% and 2% chlorohexidine. The comparison of the mechanical properties among the three groups in term of compressive strength, diametral compressive strength and microhardness showed no statistically significant differences between conventional glass ionomer and glass ionomer containing 1% or 2% chlorohexidine. Conclusion: Glass ionomers containing chlorhexidine displayed superior antibacterial activity than conventional glass ionomers without affecting the mechanical properties of glass ionomer