The objective of this in vitro study was to evaluate the long-term diffusion behavior of eugenol from different zinc oxide-eugenol mixtures through thin dentin layers. In 24 freshly extracted, caries-free human teeth standardized cavities were prepared exhibiting a dentin surface area of 7 mm2. The remaining thickness of the dentin close to the pulp chamber amounted to at least 0.2 mm. In a second experiment standardized dentin disks (0.2 mm thick) were prepared from 24 bovine incisors. The disks were embedded in epoxy resin, thus exposing a resin-free, standardized area of 7 mm2. Specimens from both experiments were inserted into a screw cap of a vial. Cp-Cap, Temp Bond, and two zinc oxide-eugenol mixtures (P/L ratios of 10:1 and 2:1) were applied to each of six specimens from both experiments. The opposing surface was in contact with 1 ml of Ringer solution. After 1 day, 7 days, and 21 days respectively, the amount of penetrated eugenol was determined with High-Performance Liquid Chromatography (HPLC). In both experiments we found a persistent release and diffusion of eugenol, irrespective of the applied material. However, for the second experiment, we saw more pronounced diffusion rates. Furthermore, the materials showed significant differences, particularly for low versus high P/L ratios.