The maize endophyte Acremonium zeae is antagonistic to kernel rotting and mycotoxin producing fungi Aspergillus flavus and Fusarium verticillioides in cultural tests for antagonism, and interferes with A. flavus infection and aflatoxin contamination of preharvest maize kernels. Chemical studies of an organic extract from maize kernel fermentations of Acremonium zeae (NRRL 13540), which displayed significant antifungal activity against Aspergillus flavus and F. verticillioides, revealed that the metabolites accounting for this activity were two newly reported antibiotics pyrrocidines A and B. Pyrrocidines were detected in fermentation extracts for 12 NRRL cultures of Acremonium zeae isolated from maize kernels harvested in Illinois (4/4 cultures), North Carolina (5/5), Georgia (1/2) and unrecorded locations within the USA (2/2). Pyrrocidine B was detected by LCMSMS in whole symptomatic maize kernels removed at harvest from ears of a commercial hybrid that were wound-inoculated in the milk stage with A. zeae (NRRL 13540) or (NRRL 13541). The pyrrocidines were first reported from the fermentation broth of an unidentified filamentous fungus LL-Cyan426, isolated from a mixed Douglas Fir hardwood forest on Crane Island Preserve, Washington, in 1993. Pyrrocidine A exhibited potent activity against most Gram-positive bacteria, including drug-resistant strains, and was also active against the yeast Candida albicans. In an evaluation of cultural antagonism between 13 isolates of A. zeae in pairings with A. flavus (NRRL 6541) and F. verticillioides (NRRL 25457), A. zeae (NRRL 6415) and (NRRL 34556) produced the strongest reaction, inhibiting both organisms at a distance while continuing to grow through the resulting clear zone at an unchanged rate. Maximum colony diameters for A. zeae (NRRL 6415) and (NRRL 13540), on potato dextrose agar after 14 d, were attained within the range of 25–30 °C, with less growth recorded at 15 ° and 37.5 ° and no growth at 5 °. Potential interactions between A. zeae and other maize endophytes are considered and the significance of these interactions relative to the aflatoxin and fumonisin contamination of preharvest maize is presented. This is the first report of natural products from Acremonium zeae.