Microorganisms often form biofilms—structured communities of microbial aggregates encased in self-produced extracellular polymeric substances (EPS). These biofilms enable adherence to surfaces and enhance microbial survival and interaction. Several plant-associated fungi, including Fusarium verticillioides, a mycotoxigenic fungus associated with maize, are known to create biofilms, necessitating research into their role in fungal persistence and disease development. This study aimed to investigate the biofilm formation capability of F. verticillioides under laboratory conditions. Our results indicated that stationary phase cultures developed a biofilm-like pellicle characterised by a cloudy, thin slime composed of hyphal aggregates. Microscopic analysis revealed a heterogeneous structure of dense, entangled hyphae alongside quantifiable EPS and extracellular DNA (eDNA) levels. The biofilms also exhibited responsiveness to factors such as pH and temperature, emphasizing their ecological relevance. Furthermore, we assessed the role of eDNA in maintaining biofilm structure through DNase treatment, which proved marginally effective in mature biofilms. This suggests complex interactions between eDNA and constituents in the EPS during maturation. The analysis of the