A phytochemical investigation on the leaves of Elsholtzia rugulosa resulted in the isolation and identification of thirty compounds, including four flavonoids (1–4), two benzene derivatives, two lignans (7 and 8), three phenylpropanoids (9–11), three phenolic acid (12–14), five sesquiterpenes (15–19), five nor-sesquiterpenes (20–24), one diterpenoids (25), and four monoterpenoids (26–30). Among of them, compounds 2–8 and 10–30 were firstly reported in the genus Elsholtzia, all compounds were originally obtained from Elsholtzia rugulosa. Furthermore, the anti-H1N1 activities of these compounds were evaluated in vitro. The results showed compounds 1, 9, 14 and 29 possessed moderate anti-influenza activity, with the inhibition rates (%) to be 49.69, 59.1, 48.21 and 44.99 at 100 μg/mL, respectively. The chemotaxonomic significance of these isolated compounds has been discussed.