In a search for naturally occurring antibacterial compounds in medicinal plants, six hitherto unknown thiophene acetylenes, named 10,11-threo-xanthopappin D, 10,11-erythro-xanthopappin D, 10,11-cis-xanthopappin B, 5-(but-4-chloro-3-hydroxy-1-ynyl)-2-(Z)-pent-3-ene-1-ynylthiophene, 5-(but-4-chloro-3-hydroxy-1-ynyl)-2-(E)-pent-3-ene-1-ynylthiophene, 5-(but-3,4-dihydroxy-1-ynyl)-2-(Z)-pent-3-ene-1-ynylthiophene and two furanosesquiterpenes, as well as fifteen known compounds, were isolated from Xanthopappus subacaulis, which has been used as a traditional Tibetan medicine in China. A biosynthetic pathway to thiophene acetylenes was proposed and, the isolated compounds were tested for their antibacterial activity against five bacteria. Within the series of thiophene acetylenes tested, 10,11-threo-xanthopappin D with a threo configuration exhibited strong activity against Bacillus subtilis, with a minimum inhibitory concentration (MIC) of 7.25μg/mL, whereas 10,11-erythro-xanthopappin D with erythro configuration possessed broad-spectrum antibacterial activity against Escherichia coli, Bacillus cereus, Staphylococcus aureus and Erwinia carotovora, with MICs of 12.5, 15.5, 7.25 and 7.25μg/mL, respectively. Meanwhile, the compounds 10,11-cis-xanthopappin B, xanthopappin B, 5-(but-4-chloro-3-hydroxy-1-ynyl)-2-(Z)-pent-3-ene-1-ynylthiophene and 5-(but-4-chloro-3-hydroxy-1-ynyl)-2-(E)-pent-3-ene-1-ynylthiophene substituted with a Cl atom at C-14 showed moderate inhibitory activity against E. coli, B. cereus, S. aureus, E. carotovora and B. subtilis, with MICs ranging from 31.25 to 62.5μg/mL. The structures of these compounds were elucidated through the comprehensive analysis of spectroscopic data, including UV, IR, MS and NMR.