Four new guaiane-type sesquiterpenes, argyin H–K (1–4), and two known analogues (5 and 6) were isolated from the leaves of Artemisia argyi Lévl et Vant. The new compounds were characterized by the basic analysis of the spectroscopic data obtained ( 1 H NMR, 13 C NMR, HMBC, and NOESY experiments), and their absolute configurations were determined by empirical approaches, combined with the exciton chirality method and electronic circular dichroism calculations. To further understand the antitumor effects of A. argyi , the antiproliferative activities of these compounds against A549, MCF-7, and HepG2 cell lines were tested in vitro using CCK-8 assays. The results showed that these compounds had significant antiproliferative effects on MCF-7, with IC 50 values of 15.13–18.63 μM, which were superior to that of oxaliplatin (i.e., IC 50 22.20 μM).