The purpose of this study was to evaluate the neuroprotective effects of omega-3 polyunsaturated fatty acid (ω3-PUFA) supplementation, alone or in combination with timolol eye drops, in a mouse model of hereditary glaucoma. DBA/2J mice (8.5-month-old) were assigned to an ω3-PUFAs + timolol, ω3-PUFAs only, timolol only, or an untreated group. Treated mice received a daily gavage administration of eicosapentaenoic acid (EPA) and docosahexaenoic acid and/or topical instillation of timolol (0.5%) once a day for 3 months. Blood was analysed regularly to determine ω3-PUFA levels and retinas were histologically analysed. Real-time PCR and Western blot were performed for retinal pro-inflammatory cytokines and macrophages. Blood arachidonic acid/EPA ratio gradually decreased and reached the desired therapeutic range (1–1.5) after 4 weeks of daily gavage with ω3-PUFAs in the ω3-PUFAs + timolol and ω3-PUFAs only groups. Retinal ganglion cell densities were significantly higher in the ω3-PUFAs + timolol (1303.77 ± 139.62/mm2), ω3-PUFAs only (768.40 ± 52.44/mm2) and timolol only (910.57 ± 57.28/mm2) groups than in the untreated group (323.39 ± 95.18/mm2). ω3-PUFA supplementation alone or timolol alone, significantly increased protein expression levels of M1 macrophage-secreted inducible nitric oxide synthase and M2 macrophage-secreted arginase-1 in the retina, which led to significant decreases in the expression levels of tumour necrosis factor-α (TNF-α). ω3-PUFA supplementation alone also resulted in significantly reduced expression of interleukin-18 (IL-18). ω3-PUFA + timolol treatment had no effect on the expression level of any of the aforementioned mediators in the retina. Supplementation with ω3-PUFAs has neuroprotective effect in the retinas of DBA/2J mice that is enhanced when combined with timolol eye drops. The continued inflammation following ω3-PUFAs + timolol treatment suggests that downregulation of IL-18 and TNF-α may not be the only factors involved in ω3-PUFA-mediated neuroprotection in the retina.