Background Oxidative stress is involved in the pathogenesis of Graves' orbitopathy (GO) and several antioxidant agents, namely, selenium, quercetin, enalapril, vitamin C, N-acetyl-l-cysteine, and melatonin, have been shown to reduce oxidative stress and its consequences in primary culture of orbital fibroblasts. In addition, selenium is effective for the treatment of mild GO. Here, we investigated the action of three additional antioxidants in orbital fibroblasts, namely, retinol, beta-carotene, and vitamin E. Methods Primary cultures of orbital fibroblasts were established from GO patients and control subjects. To induce oxidative stress, cells were treated with H2O2, after which glutathione disulfide (GSSG) (a parameter of oxidative stress), cell proliferation, hyaluronic acid, TNF alpha, IFN gamma, and IL1 beta were measured. Results H2O2-dependent oxidative stress (augmented GSSG) was associated with increased cell proliferation and cytokine release. All the three antioxidant substances reduced GSSG in both GO and control fibroblasts. beta-carotene reduced proliferation in GO, but not in control fibroblasts. IL1 beta was reduced by all three substances. Retinol reduced IFN gamma in GO and control fibroblasts. Conclusions Our study supports an antioxidant role of retinol, beta-carotene, and vitamin E in orbital fibroblasts from patients with GO and provides a basis for a possible clinical use these substances.