In this in vitro study, quantitative confocal microscopy was used to show differences in the quantity of bacteria remaining in dentin after excavation with different methods. A further parameter was the cavity volume after excavation relative to the original lesion size. Teeth with dentin caries were divided into three groups of 20 each. The caries was removed by a single operator using a slow handpiece and a round bur. In the first group, Fluorescence Aided Caries Excavation (FACE) was carried out: violet light was used to illuminate the operating field and the operator observed the cavity through a high-pass filter and removed the orange-red fluorescing areas. The second group was excavated using Caries Detector, while the third group used conventional excavation. After excavation, cavity volume was measured; samples were stained for bacteria with ethidium bromide, and they were examined using confocal microscopy under standardized conditions. The bound stain was quantified in terms of fluorescence intensity on the confocal images. Total pixel intensity was significantly lower in the FACE Group than in the Caries Detector group (p = 0.046) and in the conventional excavation group (p = 0.021). Differences in cavity volume relative to original lesion size were not statistically significant (p = 0.86, 0.35 and 0.51). Within the limitations of this in vitro study, it can be concluded that FACE is more effective in removing infected dentin without significantly increasing cavity size when compared to conventional excavation and excavation with the aid of caries detector dye.