Opportunistic fungi belonging to the Fusarium solani have become increasingly recognised as life-threatening pathogens causing keratitis and disseminated fusariosis among both healthy individuals and patients with haematological malignancies. These infections are associated with biofilm formation on different biotic and abiotic surfaces. Considering, a biofilm is a virulence factor for causing infections, the aim of this study optimising and illustrating a simple, cost-effective and highly reproducible 96 well microtitre-based method for F. solani biofilm formation via using crystal violet stain. The results revealed that the possibility of using either 570nm or 595nm as a wavelength for quantifying fungal biofilm formation. The best time for crystal violet de-staining was 10 min of incubation. This model can be used in-vitro to quantify and understand the virulence factor of fungal biofilm during infections, and for antifungal susceptibility testing.