The aim of this prospective study was to compare retinal vascular diameter measurements taken from standard fundus images and adaptive optics (AO) images. We analysed retinal images of twenty healthy subjects with 45-degree funduscopic colour photographs (CR-2 Canon fundus camera, Canon™) and adaptive optics (AO) fundus images (rtx1 camera, Imagine Eyes®). Diameters were measured using three software applications: the VAMPIRE (Vessel Assessment and Measurement Platform for Images of the REtina) annotation tool, IVAN (Interactive Vessel ANalyzer) for funduscopic colour photographs, and AO_Detect_Artery™ for AO images. For the arterial diameters, the mean difference between AO_Detect_Artery™ and IVAN was 9.1 µm (−27.4 to 9.2 µm, p = 0.005) and the measurements were significantly correlated (r = 0.79). The mean difference between AO_Detect_Artery™ and VAMPIRE annotation tool was 3.8 µm (−34.4 to 26.8 µm, p = 0.16) and the measurements were poorly correlated (r = 0.12). For the venous diameters, the mean difference between the AO_Detect_Artery™ and IVAN was 3.9 µm (−40.9 to 41.9 µm, p = 0.35) and the measurements were highly correlated (r = 0.83). The mean difference between the AO_Detect_Artery™ and VAMPIRE annotation tool was 0.4 µm (−17.44 to 25.3 µm, p = 0.91) and the correlations were moderate (r = 0.41). We found that the VAMPIRE annotation tool, an entirely manual software, is accurate for the measurement of arterial and venular diameters, but the correlation with AO measurements is poor. On the contrary, IVAN, a semi-automatic software tool, presents slightly greater differences with AO imaging, but the correlation is stronger. Data from arteries should be considered with caution, since IVAN seems to significantly under-estimate arterial diameters.