<i>Aim:</i> To establish a fluorescence threshold level that could guide a therapeutic Er:YAG laser through a caries lesion to determine a therapeutic endpoint of caries removal. <i>Materials and Methods:</i> A total of 65 extracted human teeth, 35 with dentine caries and 30 healthy, were used for this study. An Er:YAG laser system that emitted at a wavelength of 2.94 µm was used. The laser was equipped with a laser fluorescence feedback system, excitation wavelength 655 nm, to control the irradiation by the Er:YAG laser. The evaluated threshold levels of the fluorescence feedback system were 3, 7, 8, 10, 12, 15 and 20. After treatment the teeth were prepared for histological staining according to the method of Brown and Brenn for the identification of bacteria. The specimens were subjected to a quantitative evaluation of residual bacteria on the treated dentine surface. In addition, the internal fluorescence of dentine and potential fluorescence changes of dentine after laser irradiation were evaluated. <i>Results:</i> About 80% of the irradiated dentine surface showed residual bacteria with threshold levels of 20, 15, 12, and 10. Residual bacteria were not found with threshold levels of 7 and 3. The study revealed a significant increase in dentine fluorescence after laser irradiation. <i>Conclusion:</i> The results of the present in vitro study indicate that a fluorescence threshold level of 7 or 8 units can guide an Er:YAG laser to a complete removal of carious dentine.