The genus Fusarium is largely represented in fungal infections, not only mostly in plants but also in humans. Fungi belonging to the Fusarium solani species complex (FSSC) are those that are most frequently isolated in invasive fusariosis and present elevated minimum inhibitory concentrations for the main antifungal drugs used in medicine. This study is the first to investigate the resistance mechanism in FSSC by monitoring CYP51A expression in the presence of azole antifungals. After exposure to voriconazole, an overinduction of CYP51A was observed irrespective of the concentration of antifungal used and the generation studied. The same observation is made for other azole antifungals, posaconazole and tebuconazole, but not for amphotericin B. This observation could contribute to explaining why some antifungal molecules used in agriculture or medical practices may have low susceptibility for some fungi.