In an analytical study of microbial broths, the actinomycete strain Kitasatospora sp. P07101 was found to produce three new congeners, which were designated hazimycins B (1), C (2), and D (3), together with the previously reported hazimycin (renamed hazimycin A (4)). The structures of these hazimycins were examined using various spectroscopic methods including nuclear magnetic resonance (NMR), and the results revealed that 1-3 were analogues of hazimycin with the replacement of one of the two isonitrile groups in 4 by an NH-formyl group in 1, the two isonitrile groups and an amide group by two NH-formyl groups and a nitrile group in 2, and the two isonitrile groups and two amide groups by two NH-formyl groups and two nitrile groups in 3. Only hazimycin A exhibited moderate antimicrobial activities against Gram-positive bacteria and Candida albicans. These results indicated that the presence of two isonitrile groups in the hazimycin structure is essential for antimicrobial activity.