Considering the effect of cavity disinfecting agents on the bonding and sealing ability of restorations bonded to dentin, the aim of this study was to evaluate the effect of chlorhexidine (CHX) disinfecting agent on the marginal gaps of Cl V giomer restorations. Cl V cavities were prepared on the buccal surfaces of 60 sound bovine permanent incisors in this in vitro study, with the occlusal and gingival margins in enamel and dentin, respectively. The teeth were randomly divided into two groups (n=30). The teeth in groups 1 and 2 were restored without and with the use of the disinfecting agent in the cavity, respectively, before applying the adhesive. BeautiBond one-step self-etch adhesive and Beautifil II giomer were used to restore the cavities in both groups. After thermocycling and sectioning of the samples, the sizes of marginal gaps at gingival margins were measured in µm under a stereomicroscope. Mann-Whitney U test was used to compare marginal gaps at P<0.05 level of significance. The means of marginal gaps were significantly different between the two study groups (U=180, P<0.001), with higher means of marginal gaps in group 2 (with CHX disinfection) compared to group 1 (without CHX disinfection) (P<0.0005). Application of CHX for the disinfection of cavities in giomer restorations resulted in an increase in gingival margin gaps.