Twenty-one known specialized metabolites were isolated from the flowers of Vernonanthura nudiflora (Less.) H. Rob., the structures of the compounds were established based on 1D and 2D NMR spectroscopic experiments. Another 28 compounds were putatively identified using the dereplication technique by UHPLC-HRMS/MS. Twenty-three of the compounds are being reported for the first time in this species. The sesquiterpene lactone mixture piptocarphins A and B ( 17 + 18 ), and the flavone velutin ( 14 ) were tested against various microorganisms and showed promising activity against Mycobacterium tuberculosis with MICs of 15.6 μg/mL and 31.2 μg/mL, respectively. Furthermore, 17 + 18 showed higher cytotoxicity against VERO cells (IC50 = 7.0 ± 1.73) compared to compound 14 (IC50 = 85.0 ± 10.6 µg/mL). These findings reveal the feasibility of utilizing UHPLC-ESI-HRMS/MS-based dereplication strategy on complex fractions to identify specialized metabolites, and that V. nudiflora flowers constitute a source of compounds with antimycobacterial potential.