Five new antibacterial ambiguine K-O isonitriles (1-5) and eight previously described indole alkaloids were isolated from the cultured cyanobacterium Fischerella ambigua (UTEX 1903) by bioassay-guided fractionation. The planar structures of the new compounds were determined by spectroscopic analysis including MS and 1D and 2D NMR. X-ray crystallography was used to determine the absolute stereoconfiguration of ambiguine K isonitrile. The isolates were evaluated for their antibacterial activities against a set of bacterial targets, including Mycobacterium tuberculosis and Bacillus anthracis. Ambiguine K and M isonitriles showed the most potent activity against M. tuberculosis, with MIC values of 6.6 and 7.5 microM, respectively. Ambiguine A isonitrile showed the most potent activity against B. anthracis, with a MIC of 1.0 microM.