Sudden death syndrome (SDS) caused by Fusarium virguliforme is among the most important diseases affecting soybean yield in the USA. The exploitation of biocontrol agents (BCAs) can be a valuable resource to suppress F. virguliforme populations. Trichoderma spp. are known to be antagonistic against a broad range of phytopathogens, however their activity against F. virguliforme has not been fully explored. Therefore, the objective of this study was to screen native Trichoderma isolates against F. virguliforme in-vitro and evaluate the performance of highly antagonistic Trichoderma isolates under semi-field conditions (microplot) using two application methods; seed-coating and in-furrow. A total of 41 Trichoderma isolates were screened in a dual plate assay. The most effective isolates belonged to the T. harzianum species and were able to inhibit F. virguliforme radial growth by up to 92%. Three selected isolates were tested in microplots. The isolates showed a protective effect in both application methods by significantly reducing root rot and disease index 30 days after planting, compared to the plants inoculated with only F. virguliforme. Further, a qPCR assay showed an increased extent of Trichoderma colonization of soybean roots when the BCAs were delivered in-furrow. Our results highlight the potential of native Trichoderma isolates to inhibit F. virguliforme growth and reduce SDS severity on soybean.