Activity-guided fractionation based on topoisomerase I inhibitory activity lead to the isolation of ten lignans (1—10) from the methylene chloride extract of the bark of Machilus thunbergii SIEB. et ZUCC. (Lauraceae). These were identified as machilin A (1), erythro-austrobailignan-6 (2), meso-monomethyl dihydroguaiaretic acid (3), meso-dihydroguaiaretic acid (4), galbacin (5), machilin F (6), nectandrin A (7) nectandrin B (8), (−)-acuminatin (9) and (7S,8S)-7-(4-hydroxy-3-methoxyphenyl)-1′-formyl-3′-methoxy-8-methyldihydrobenzofuran (10) by spectral evidence. In DNA topoisomerase I and II assays in vitro at a concentration of 100 μM, 4 showed the most potent inhibitory activity, 93.6 and 82.1% inhibition, respectively, and 8 showed 79.1 and 34.3% inhibition, respectively. All of these compounds exhibited weak or no cytotoxicities against either the human colon carcinoma cell line (HT-29) or the human breast carcinoma cell line (MCF-7).