This study was aimed at determining the ability of different coronal temporary fillings to prevent corono-apical penetration of bacteria. A total of 103 human teeth, including three controls, were instrumented, obturated with gutta-percha, coronally sealed with either Cavit, Intermediate Restorative Material (IRM), glass-ionomer cement, Cavit/glassionomer cement, or IRM/glass-ionomer cement, respectively. Each root was fixed with wax between two chambers: the coronal chamber harboring soy broth with 108 colony-forming units of Streptococcus mutans/ml, the apical chamber containing sterile soy broth. The latter was checked daily for turbidity, indicating corono-apical penetration of bacteria. The Cavit group, the IRM group, and the Cavit/glassionomer cement group showed significantly more leakage than the glass-ionomer cement group of the IRM/glass-ionomer cement group. If a sample leaked, all except one (IRM/glass-ionomer cement) leaked before day 12. This in vitro study seems to indicate that only glass-ionomer cement and IRM combined with glass-ionomer cement may prevent bacterial penetration to the periapex of root-filled teeth over a 1-month period.