Cyclodextrins (CDs) are widely used compounds in pharmaceutical industry. They enhance solubility, bioavailability and stability of many drugs. Recently, several of CD derivatives have been synthetized in order to improve their physicochemical properties and inclusion capacities. Based on their pharmaceutical importance, many studies demonstrated the activity of CDs in drug complexation, however, there is limited information available about their cytotoxic effects. The aim of our study was to investigate the cytotoxic properties of various CD derivatives. We performed MTT cell viability assays on the Caco-2 human colon carcinoma cell system. In addition, we investigated cholesterol-CD complexation by an HPLC method, which determined the cholesterol content of the cholesterol-CD complex. The viability tests showed significant differences between the cytotoxicity of the CD derivatives. Cell toxicity of methylated CDs decreases DIMEB>TRIMEB>RAMEB. The anionic carboxymethylated derivative (CMBCD) and cationic quaternary amino beta-cyclodextrin (QABCD) proved to be less toxic than the methylated ones. Most of the second generation CD derivatives, which contain ionic substituents beside the methyl groups, showed less cytotoxicity than the parent compounds, only succinyl random methylated beta-CD (SU-RAMEB) and RAMEB represent the similar toxicological properties on Caco-2 cells. Harmful attributes of RAMEB, DIMEB and their cholesterol complexes were also investigated in our in vitro system. RAMEB and DIMEB cholesterol complex derivatives showed slight cytotoxic effects compared to the parent compounds. In conclusion, our studies demonstrated a significant correlation between the cytotoxic effect and the cholesterol complexation attributes of CD derivatives.