Background: The aim of this study was to assess the expression of cytokines/chemokines in tears from patients with non-advanced primary open-angle glaucoma and patients with non-severe dry eye disease versus healthy controls. Methods: This prospective, observational cohort study enrolled patients with confirmed or suspected non-advanced primary open-angle glaucoma who received any prostaglandin analogue monotherapy for longer than 6 months, patients with non-severe dry eye disease, and healthy controls. Expression of interleukin-1β, interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin-8, interleukin-10, and interleukin-12; tumor necrosis factor α; vascular endothelial growth factor; granulocyte-macrophage colony-stimulating factor; and interferon γ was assessed. Results: 107 participants were enrolled (primary open-angle glaucoma, n = 41; dry eye disease, n = 30; and healthy controls, n = 36). Compared with healthy controls, interleukin-6 was significantly higher ( p = 0.0001) in patients with primary open-angle glaucoma and interleukin-1β ( p = 0.0144), interleukin-6 ( p < 0.0001), and interleukin-10 ( p = 0.0392) were higher in patients with dry eye disease. Compared with patients with dry eye disease, patients with primary open-angle glaucoma had significantly lower levels of interleukin-4 (21.79 vs 20.18 pg/mL; p = 0.0012) and significantly higher levels of vascular endothelial growth factor (367.75 vs 609.28 pg/mL; p = 0.0058), tumor necrosis factor α (14.27 vs 17.93 pg/mL; p = 0.0048), and interleukin-6 (17.95 vs 27.48 pg/mL; p = 0.0145). In patients with primary open-angle glaucoma, interleukin-1β expression ( p = 0.0011) was lower than in those who received intraocular pressure–lowering eye drops without preservatives compared with those who received eye drops with preservatives. Conclusion: Different cytokine/chemokine expression profiles in tears of patients with primary open-angle glaucoma and dry eye disease strongly suggest the involvement of a variety of signaling pathways in the pathogenesis of these ophthalmic processes.