While much is known about how Fusarium graminearum initiates disease on host plants, there are still knowledge gaps related to mechanisms that increase success in infection. To better understand early infection, we focused on two aspects of the plant-pathogen interaction. Biofilms are three-dimensional formations important to many microbes for protection from adverse environmental conditions. Biofilms have primarily been studied in single-celled organisms, but there is a growing body of work on filamentous fungal biofilms. Biofilm formation is initiated in vitro with the adhesion of propagules to a surface, followed by growth of the structures and development of an extracellular matrix, then dispersal of propagules and senescence of biofilms. We profiled the transcriptome of biofilm formation over time, and characterized genes significant to this process. Aquaporins are channel proteins that bring molecules dissolved in water into cells. In F. graminearum, aquaporins are important in growth, development, and spore formation. Knockout mutants of these genes and others identified by transcriptomics, produced altered biofilms, especially in matrix composition. We have identified novel early infection characteristics in F. graminearum through characterization of the biofilm in vitro and in planta.