β-cyclodextrin (β-CD) is a suitable excipient for peroral use, which improves the solubility of lipophilic drugs, as well as for colon-specific drug release when it is mixed with coating polymers. The first aim of this work was to examine the suitability of various enzymes as a simple in vitro model for the glycolytic activity in the human colon. α-Amylase (source Aspergillus oryzae) and taka diastase (source A. oryzae) showed remarkable degradation capacity of free β-CD, whereas other α-amylases (sources Bacillus subtilis or Hog pancreas) were found to be unsuitable. The next aim was to find out if film-incorporated β-CD is also degraded by these enzymes. Therefore, diffusion studies of 5-aminosalicylic acid (5-ASA) through Eudragit® RS or Eudragit® NE films containing β-CD were performed with taka diastase present in the buffer medium. Pronounced diffusion of the drug through the Eudragit® RS film was found only when swelling excipients like crosslinked sodium carboxymethylcellulose (CMC-CL sodium) or polyvinylpyrrolidone (PVP 25) were present in the film, indicating enhanced accessibility of β-CD by the enzyme. Films containing CMC-CL without β-CD showed even higher permeability, which also points to enzymatic degradation of CMC-CL. Permeabilization by taka diastase of Eudragit® NE films without swelling agents correlated with the β-CD content, whereas control films containing talcum remained impermeable upon enzyme action. Furthermore, the β-CD degradation capacity of colonic bacteria like Escherichia fergusonii, Serratia odorifera or Proteus mirabilis was examined with β-CD coatings on tablets, which contained bisoprolol as a model drug. Tablets with β-CD-containing Eudragit® RS coatings showed the highest drug release upon incubation with P. mirabilis. The moderate drug release by E. fergusonii could be increased almost to the same level when the bacteria were pre-incubated for 24 h in medium containing 2.5 mg/ml β-CD, indicating the induction of glycolytic enzymes by β-CD in this colonic bacteria strain.