The ability of two Aspergillus flavus Link isolates (CT3 and K49) to reduce aflatoxin contamination of corn was assessed in a four-year field study (2001 to 2004). Soil was treated with six wheat inoculant treatments: toxigenic isolate F3W4; the non-toxigenic isolate K49; the non-aflatoxigenic isolate CT3, two mixtures of CT3 or K49 with F3W4; and an autoclaved wheat control, applied at 20 kg/ha. In 2001, inoculation with the toxigenic isolate increased corn grain aflatoxin levels by 167% compared to the non-inoculated control, while CT3 and K49 inoculation reduced aflatoxin levels in corn grain by 86% & 60%, respectively. In 2002, inoculation of CT3 and K49 reduced aflatoxin levels by 61% and 76% compared to non-inoculated controls, respectively. In 2001 mixtures of toxigenic and non-toxigenic isolates had little effect on aflatoxin levels, but in 2002 inoculation with mixtures of K49 and CT3 reduced aflatoxin levels 68 and 37% compared to non-inoculated controls, respectively. In 2003 and 2004, a low level of natural aflatoxin contamination was observed (8 ng/g). However, inoculation with mixtures of K49+F3W4 and CT3+F3W4, reduced levels of aflatoxin 65 to 94% compared to the toxigenic strain alone. Compared to the non-sclerotia producing CT3, strain K49 produces large sclerotia, has more rapid in vitro radial growth, and a greater ability to colonize corn when artificially inoculated, perhaps indicating greater ecological competence. Results indicate that non-toxigenic, indigenous A. flavus isolates, such as strain K49, have potential use for biocontrol of aflatoxin contamination in southern U.S. corn.