In order to find key intermediates in the transformation of emestrin (2) to its degradation products (3, 4, and/or 5), further investigation of the mycelial acetone extract of Emericella striata was performed. A new compound designated emestrin B (1) (from the fraction slightly more polar than emestrin) and shamixanthone (from the fraction slightly less polar than emericellin) were isolated. The structure of emestrin B (1), including its absolute configuration, was established on the basis of spectroscopic investigations (FD mass spectrometry, 1H-NMR, 13C-NMR, circular dichroism, etc.). Emestrin B (1) is an epitrithio derivative corresponding to the antifungal macrocyclic epidithiodioxopiperazine emestrin (2). Preliminary test showed emestrin B (1) exhibited inhibitory activity at 25 μg per disc against Bacillus subtilis and Escherichia coli. Further studies on the antibacterial and antifungal activity are planned.