Background: The aim of this study was to assess the effect of incorporation of zinc oxide nanoparticles (ZnO-NPs) to glass-ionomer cement (GIC) (Fuji II SC, GC Corp., Tokyo, Japan) on subgingival accumulation of mutans streptococci and lactobacilli under orthodontic bands.Materials and Methods: In order to conduct this in vivo split-mouth study, 20 patients aged between 7 and 10 years who required lingual holding arch on their mandibular first molars were divided into two groups. In one group, Fuji II SC GIC was used for cementation of the right molar band, and the same cement containing 2 wt% ZnO-NPs was used for the left one. The opposite was performed for the second group while the operator was blinded to the cement types. Subgingival microbial sampling was performed 16 weeks after cementation of lingual arch. Mutans streptococci and lactobacilli colony counts were compared. Paired t-test was used to compare the two cement groups. Data were analyzed using SPSS version 21, and P ≤ 0.05 was considered statistically significant.Results: The mean colony counts of mutans streptococci, lactobacilli, and total bacterial count in Fuji II SC containing ZnO-NPs were significantly lower than the corresponding values in plain Fuji II SC group.Conclusion: Incorporation of ZnO-NPs into GIC reveals antimicrobial features against mutans streptococci and lactobacilli under orthodontic bands.