Pleurotus tuber-regium has been reported to lower dexamethasone-induced ocular hypertension. Effects of P. tuber-regium and 0.005% latanoprost on the circadian intraocular pressure (IOP) cycle in cats were investigated for a possible phase difference. The research was conducted in three phases. In phase 1, the baseline diurnal IOP of 12 cats was determined. In phase 2, the cats were divided into two groups (X and Y). Group Y was left untreated, while group X was treated with 0.1% dexamethasone topically. In phase 3, group X was further divided into two subgroups (X1 and X2). One eye of each cat in subgroup X1 was treated with 0.005% latanoprost; at the same time, one eye of each cat in subgroup X2 received P. tuber-regium topically. IOP was measured at 3-h intervals for 24 h, after 2 weeks of treatment. The mean baseline IOP was 12.8 mm Hg. After 2 weeks, both P. tuber-regium and latanoprost significantly (P < 0.0001) reduced ocular hypertension induced (mean of 23.2 mm Hg) in the right eye of subgroups X1 and X2 to 15.6 and 14.4 mm Hg, respectively. Their mean dark phases were 15.8 and 14.2 mm Hg, while their mean light phases were 15.5 and 14.6 mm Hg, respectively. Control group Y had a mean IOP of 13.2 mm Hg. Both P. tuber-regium and latanoprost lower dexamethasone-induced ocular hypertension significantly. While P. tuber-regium is more effective in the light phase, latanoprost is more effective in the dark phase; however, the difference is not significant. This work further confirms P. tuber-regium as a candidate to study for a source of drug that could be useful in IOP management in clinical practice.