Oxymatrine is the primary pharmacological component of Sophora flavescens Ait. In the present study, we investigated the protective effect of oxymatrine against Coxsackievirus B3-induced myocarditis in mice. Coxsackievirus B3-infected HeLa cells were treated with oxymatrine and the viral titer, as well as the degree of cellular proliferation were determined. Additionally, BALB/c mice were infected with Coxsackievirus B3 and received differing concentrations of oxymatrine. On days 5 and 12 following treatment, mice were sacrificed, and serum lactate dehydrogenase, creatine kinase-MB isozyme, and tumor necrosis factor-α levels were quantified. The heart index and degree of myocardial tissue inflammation were also assessed. On day 5, the Coxsackievirus B3 TCID50 values of the heart tissue, and the expression of NTR, IFN-γ, and TNF-α genes in the myocardial tissue were measured. Our results showed that oxymatrine exhibits potent antiviral effects on Coxsackievirus B3 as 50% inhibition was achieved at a concentration as low as 0.238 mg/mL. Oxymatrine markedly reduced the viral titer and inhibited cardiac myocyte pathology exhibited in viral myocarditis. Furthermore, oxymatrine treatment reduced the expression of Coxsackievirus B3 NTR and mouse TNF-α genes compared to the controls. Therefore, our findings indicate that oxymatrine is a promising potent antiviral agent against Coxsackievirus B3-induced myocarditis.