Brenda Wingfield's research focuses on the speciation and evolution of fungi, predominantly non-model Ascomycetes. This includes research on genetic variation within and between species, and a component of her work is considered molecular taxonomy. She has an interest in basic evolutionary biology focused on ribosomal RNA genes, mycoviruses, and mating type genes. Wingfield collaborates with research groups working on a spectrum of different organisms, primarily in association with the tree pathology research group and the DST/NRF Centre of Excellence in Tree Health Biotechnology, emphasizing important plant and tree pathogens. She is also involved with the Grain Research Programme (GRP) at FABI, which focuses on fungal pathogens of agricultural crops.

Understanding the population diversity of pathogens is crucial for controlling disease spread. The genetic diversity of a pathogen is determined not only by the fungal genome but also by extra chromosomal nucleic acids, particularly mycoviruses which have been associated with hypovirulence in some fungi. Wingfield's research investigates the presence of viruses in South Africa's significant tree pathogens, with the aim of using these as a means of biological control.

She holds the DST/NRF SARChI chair in Fungal Genomics. Advances in sequencing fungal genomes allow her team to explore questions previously unattainable. Current genomics research focuses on Fusarium species related to the pine pathogen Fusarium circinatum and the genus Ceratocystis. Wingfield's team sequenced Africa's first fungal genome and has several re-sequencing projects aimed at developing diagnostics for the forestry industry. Additionally, they aim to understand how these species cause disease using a systems biology approach.