Although several studies have shown that chlorhexidine (Cx) has bactericidal activity and exerts toxic effects on periodontal tissues a few studies evaluated mechanisms to reduce its adverse effects maintaining the antimicrobial properties. Therefore, the aim of the present study was to investigate the in vitro antimicrobial activity and cellular cytotoxicity of Cx included on cyclodextrins (Cd), α, β or Hp-β-cyclodextrins (Hp-β-Cd). The influence of Cds was determined by increasing its molar rate 1:1 to 1:4 in relation with free Cx. The minimal inhibitory concentrations (MICs) for Candida albicans, Aggregatibacter actinomycetemcomitans actinomycemcomitans and Streptococcus mutans were determined. An ergosterol solubilization assay was carried out using the C. albicans model and osteoblasts, fibroblasts and tumoral Caco-2 cells for cytotoxicity assay. The antimicrobial activity results in a significant growth inhibition of C. albicans when it was treated with Cx:α-Cd complexes, whereas Cx:β-Cd was more effective for A. actinomycetemcomitans, and Cx:Hp-β-Cd complexes was for S. mutans when compared to the other complexes. The cytotoxicity for fibroblasts and osteoblasts decreased in relation with each kind of Cd been β-Cd ≤ Hp-β-Cd ≤ α-Cd. Although the Hp-β-Cd inclusion complexes had more severe effects on Caco-2 cells, all complexes exhibited less cytotoxicity than free Cx. The α-Cd, β-Cd and Hp-β-Cd increase the antimicrobial activity of Cx, but decrease its cytotoxic effects on mammalian cells. Taken together these findings suggest that cyclodextrins are a tool for modulation of effects of Cx. It could be useful to design Cx/Cd delivery systems with high efficacy and minimum cytotoxic effects.