The aim of this study was to determine the role of rubber dam isolation and the influence of salivary contamination during cavity preparation on the collection of samples of dentine for microbiological examination. Cavities were prepared in 23 carious teeth. Access to caries was gained and a sample of soft, stained demineralized tissue was taken with a round bur. After removing the rubber dam each cavity was flooded with saliva and after thorough washing and drying a second dentine sample was taken. Cavity preparation was completed and the cavity flooded with saliva. After thorough washing and drying a final sample was taken. A data set from another study served as a control for bacterial contamination at the end of cavity preparation where a rubber dam remained in place throughout. There were no significant differences in the numbers of bacteria in dentine samples gathered with and without rubber dam, which implies that salivary contamination can be removed by thorough washing and drying of the cavity.