A new cytotoxic indole alkaloid containing an isonitrile group, brasilidine A (1), has been isolated from the actinomycete Nocardia brasiliensis IFM 0089 and the structure elucidated on the basis of its spectroscopic data. The molecular formula of brasilidine A (1) was established to be C17H16N2 by HREIMS, and an IR band at 2105 cm-1 indicated the presence of an isonitrile group. UV absorptions [λmax (CH3CN) 343 (33 000), 270 (33 000), and 226 nm (78 000)] were characteristic of a 3-alkylindole chromophore. The 1H and 13C NMR spectra, along with 1H-1H COSY, HMBC, and NOESY data, revealed the structure including a 3-substituted indole ring and a triene unit conjugated with an isonitrile group. Brasilidine A (1) is a new indole alkaloid with a unique triene unit conjugated with an isonitrile group. It exhibited cytotoxicity against several tumor cell lines in vitro, including multidrug-resistant cell lines P388/ADM (IC50, 0.56 µg/mL) and CHO/MDR (IC50, 3.43 µg/mL). It also showed inhibitory activity against some Gram-positive bacteria and fungi, especially Aspergillus niger (MIC, 0.39 µg/mL) and Mycobacterium smegmatis (MIC, 0.78 µg/mL).