Aims: To evaluate retinal nerve fiber layer (RNFL) thickness in eyes with Graves’ orbitopathy (GO), in eyes with ocular hypertension (OHT) and in a control group of healthy eyes. Methods: Observational, controlled cross-sectional study. We evaluated all patients with primary open-angle glaucoma (POAG) and all patients with GO and intraocular pressure >23 mm Hg in primary position examined from March 2006 to June 2007. Forty apparently healthy patients (80 eyes) were enrolled as a control group. Complete ophthalmic evaluation, visual field (VF) examination with the Humphrey Visual Field Analyzer and RNFL thickness measurement with optic nerve tracking optical coherence tomography (ONT-OCT; OCT/SLO, OTI, Toronto, Ont., Canada) were performed. Results: Among 116 eyes with POAG [58 patients, 32 males, 26 females, mean age 62 (46–71) years], RNFL was reduced in 87 eyes (75%, p = 0.05) when compared with the control group, and a good correlation was found between RNFL thickness and VF abnormalities (Spearman’s ρ 0.822; p = 0.001). Among 60 eyes [30 patients, 12 males, 18 females, mean age 56 (50–69) years] with GO and OHT, nonglaucomatous diffuse abnormalities of the VF were detected in 44 eyes (73.3%, p = 0.03), while RNFL thinning was present in 14 eyes (9 patients, 23.3%, p = 0.03). No correlation was found between RNFL thickness and VF abnormalities (Spearman’s ρ 0.365; p = 0.02). No significant differences in RNFL pattern were present between the group with GO, OHT and RNFL thinning and the group with POAG. Conclusions: In patients with GO and OHT, evaluation of RNFL thickness with ONT-OCT may represent an objective diagnostic technique for detecting optic neuropathy.