In this study, we investigated the effects of Perilla frutescens var. acuta aqueous extract (PFA) on eyestrain using in vitro, ex vivo, and clinical tests. We confirmed the antioxidant activity of compounds present in the extract by using high-performance liquid chromatography. PFA contains antioxidant compounds such as rosmarinic acid, luteolin-7-O-glucuronide, and apigenin-7-O-glucuronide, and has been shown to inhibit reactive oxygen species generation in C2C12 muscle cells. In an ex vivo study, PFA inhibited the carbachol (100 μM)-induced contraction of ciliary muscle from rabbit eyeball. We then investigated the mechanism by which PFA caused muscle relaxation and found that it effectively increased cyclic GMP production and inhibited PDE5A activity, but did not affect the cyclic AMP pathway, in cultured human aortic smooth muscle cells. In addition, PFA (50, 100, and 200 µg/mL) dose-dependently decreased basal intracellular calcium in smooth muscle cells and attenuated the endothelin-1-stimulated increase in intracellular calcium. Finally, we performed a clinical study to evaluate the effects of PFA on the near point of accommodation (NPA) after visual display terminal (VDT) work. Thirty participants were randomized to either PFA (500 mg/day) or placebo, and intake for one consecutive week. NPA was evaluated before and after 2 h of VDT work, and was found to be improved in the PFA-treated group. Thus, our findings show that PFA may ameliorate visual fatigue.