This study aimed to investigate the null hypothesis that there is no relationship between the microhardness of carious dentine and its native autofluorescence (AF). Six extracted, carious molars were sectioned through natural lesions in the mesio-distal longitudinal plane. The Knoop microhardness (Knoop hardness number, KHN) of the cut surfaces of each sample was recorded at regular intervals through sound and carious dentine. Confocal fibre-optic micro-endoscopic (CFOME) examination of the carious dentine and the sound dentine was carried out at the same intervals using the Cellvizio system (600 microm wide, flat-end probe) with an excitation wavelength of 488 nm. The blindly collected numerical data were analysed using the original microhardness KHN. The data analysis indicated that the autofluorescence signals increased significantly when the microhardness of dentine dropped below 25 KHN. Therefore, the null hypothesis was disproved, and it was concluded from this investigation that the autofluorescent signal intensity recorded using CFOME could produce an objective and reproducible correlation to the microhardness of carious dentine. Confocal fibre-optic micro-endoscopic examination could have clinical potential as a technology to help delineate the carious dentine that might be excavated in a clinical procedure in vivo.