Micacocidin (3), a Zn-free derivative of micacocidin A (1), was prepared to evaluate its antimicrobial activity in comparison with 1 and to obtain a starting material for chemical modification of 1. The structure of 3, quite unlike those of any previously known antimicrobial agents, was elucidated by 1-D and 2-D homonuclear and heteronuclear NMR and mass spectroscopy. Micacocidin (3) thus prepared exhibited weak or no antibacterial activity except against Mycoplasma species, i.e. 3 showed stronger activity than 1. It is noteworthy that 3 displayed high activity against fungi such as Candida, Aspergillus and Trichophyton species.