Pyroligneous acids (PA) from hardwood, softwood, and bamboo significantly disinfected encephalomyocarditis virus (EMCV). Twenty-five kinds of phenolic derivatives in the PAs were identified and quantified. The total amounts of phenolic compounds in bamboo PA is higher than those in the PAs from softwood and hardwood. Phenol, 2-methoxyphenol, 2-methoxy-4-methylphenol, and 2-methoxy-4-ethylphenol are the most abundant compounds in the PAs examined. The activities of all the phenolic compounds against the encephalomyocarditis virus were assessed. The number of phenolic hydroxyl groups significantly affects the antiviral activity, and catechol and its derivatives exhibit higher viral inhibition effects than other phenolic derivatives. In addition, substituents affect the antiviral activity of the compounds. Phenolic compounds with a methyl group show higher activities than with a methoxyl group (e.g., 2-methylphenol > 2-methoxyphenol). Moreover, the relative position of functional groups also plays a key role...