Background To evaluate the effect of a dietary combination of omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) compared to single PUFA supplementations on the outcome of a substantial elevation of intraocular pressure (IOP) in rats. Methods Sprague Dawley rats were fed for 6 months with either a control diet, a diet enriched with omega-3 PUFAs (eicosapentaenoic acid, EPA, and docosahexaenoic acid, DHA), a diet enriched with omega-6 PUFAs (γ-linolenic acid, GLA) or a diet enriched with both omega-3 and omega-6 PUFAs (EPA + DHA and GLA). After 3 months of feeding, elevation of IOP was induced by photocoagulation of the episcleral veins, limbus and trabecular meshwork using a 532-nm laser. IOP and scotopic electroretinograms (ERGs) were monitored after the induction of IOP elevation until the end of the nutritional supplementation. Retinal morphometry and GFAP immunohistochemistry were performed 3 months after laser photocoagulation. Retinal ganglion cells (RGCs) were quantified using retrograde labelling. Results A significant rise in IOP was observed in the laser-treated eyes. PUFA supplementation did not influence the time course of IOP in the laser-treated eyes. Three months after laser photocoagulation, the activation of glial cells observed in the laser-treated eyes was significantly lower in animals fed with the EPA + DHA + GLA diet when compared to those fed the control diet, while single supplementations with either EPA + DHA or GLA were not effective. The same protective effect of the EPA + DHA + GLA combination was observed on retinal structures in the laser-treated eyes. However, PUFA supplementation did not influence either ERG b-wave amplitude or the RGC loss in the laser-treated eyes. Conclusions This study demonstrates that a 6-month supplementation with a combination of omega-3 and omega-6 PUFAs is more effective than single supplementations, since the EPA + DHA + GLA dietary combination prevented retinal cell structure and decreased glial cell activation induced by the elevation of IOP in rats.