Purpose: To compare the antifibrotic effect of vitamin E isoforms α-, γ-, and δ-tocotrienol on human Tenon's fibroblasts (hTf) to the antimetabolite mitomycin C. Methods: Antifibrotic effects of α- (40, 60, 80, 100, and 120μM), γ- (10, 20, 30, and 40μM) and δ-tocotrienol (10, 20, 30, and 40μM) on hTf cultures were evaluated by performing proliferation, migration and collagen synthesis assays. Whereas for vitamin E the exposure time was set to 7days to mimic subconjunctival application, cultures were exposed only 5 min to mitomycin C 100μg/ml to mimic intraoperative administration. Cell morphology (phase contrast microscopy) as an assessment for cytotoxicity and cell density by measuring DNA content in a fluorometric assay to determine proliferation inhibition was performed on day 0, 4, and 7. Migration ability and collagen synthesis of fibroblasts were measured. Results: All tested tocotrienol isoforms were able to significantly inhibit hTf proliferation in a dose-dependent manner (maximal inhibitory effect without relevant morphological changes at day 4 for α-tocotrienol 80μM with 36.7% and at day 7 for α-tocotrienol 80μM with 42.6% compared to control). Degenerative cell changes were observed in cultures with concentrations above 80μM for α- and above 30μM for γ- and δ-tocotrienol. The highest collagen synthesis inhibition has been found with 80 µM α-tocotrienol (62.4%) and no significant inhibition for mitomycin C (2.5%). Migration ability was significantly reduced in cultures exposed to 80 µM α- and 30 µM γ-tocotrienol (inhibition of 82.2% and 79.5%, respectively, compared to control) and also after mitomycin C treatment (60.0%). Complete growth inhibition without significant degenerative cell changes could only be achieved with mitomycin C. Conclusion: In vitro, all tested tocotrienol isoforms were able to inhibit proliferation, migration and collagen synthesis of human Tenon's fibroblasts and therefore may have the potential as an anti-scarring agent in filtrating glaucoma surger