The potential of Microsphaeropsis sp. (isolate P130A) as an antagonist of Gibberella zeae was tested under in vitro and field conditions. Firstly, an in vitro method of ascospore production was developed on wheat and corn residues. The plant type (corn or wheat), residue type (straw/stalk or grain), and incubation conditions (closed or open) had a significant effect on ascospore production. Perithecia were more abundant on wheat and corn grain incubated under open conditions. On these two substrates, the application of Microsphaeropsis sp. significantly reduced ascospore production. On wheat, the antagonist had a significant effect when applied 2 weeks before (-2), at the same time (0), and 4 weeks after (+4) inoculation with G. zeae, with 1.73, 0.31, 1.11, and 1.36 log ascospores per cm2 for the control, -2, 0, and +4 weeks treatments, respectively. On corn, Microsphaeropsis sp. had a significant effect when applied 2 weeks before, at the same time, 4 weeks after, and 6 weeks after inoculation with G. zeae with 3.02, 0.23, 1.29, 2.35, and 2.22 log ascospores per cm2 for the control, -2, 0, +4, and +6 weeks treatments, respectively. When applied to crop residues in the field as postharvest or preplanting applications, Microsphaeropsis sp. had no effect on the pattern of perithecia maturation, but significantly reduced the number of perithecia produced on two sampling dates, May 1998 and July 1999. There is a potential to biologically reduce the initial inoculum of G. zeae; however, more work is needed to optimize the efficacy of the biocontrol agent.