Circadian rhythms are biological processes that occur in approximately 24-hour cycles in organisms. Disruption of circadian rhythms (CD) is thought to have adverse effects on many organs, including the eyes. The aim of this study was to evaluate the effects of circadian disruption on the cornea and conjunctiva, as well as the impact of two forms of omega-3 supplementation on the ocular surface, using a rat model of circadian disruption. This experimental study included 32 female Wistar albino rats, which were divided into four groups. The control group (Group 1) was maintained under normal feeding and sleeping conditions without any disruption. Circadian disruption was induced in the other three groups, which were administered saline (Group 2), fish oil (Group 3), or flaxseed oil (Group 4) via oral gavage. On the 31st day, all rats were euthanized, and corneal, conjunctival, and palpebral tissues were collected from both eyes through enucleation. Histological examination and immunohistochemical analysis of Caspase-3 (Cas-3), tumor necrosis factor-alpha (TNF-α), and PERIOD-2 (PER-2) were performed on the corneal and conjunctival tissues. Group 2 exhibited significantly thicker corneas compared to Group 1 (P < 0.001). Additionally, hyperemia, inflammatory cell infiltration in the conjunctiva, higher expression levels of Cas-3 and TNF-α, and decreased PER-2 expression were observed in the corneal and conjunctival tissues of Group 2. These pathological changes were minimal or absent in Groups 3 and 4. Notably, Group 3 showed better amelioration of these alterations compared to Group 4. Disruption of circadian rhythms can have a negative impact on the cornea and conjunctiva. Omega-3 supplementation demonstrated a significant protective effect against ocular tissue damage induced by circadian disruption. Fish oil was more effective than flaxseed oil in reducing corneal thickening, inflammation, and apoptotic marker expression, highlighting its potential as a therapeutic intervention for circadian rhythm-related ocular pathologies.