Patients with aspergillosis have a poor prognosis because there are no effective antifungal agents except for amphotericin B. However, administration of amphotericin B is limited in immunocompromised patients due to its toxicity, which includes impairment of renal function. We attempted to evaluate the efficacy of a sequential combination of antifungal agents against Aspergillus fumigatus strain. The in vitro effects of sequential combinations of antifungal agents against five strains of Aspergillus fumigatus were determined by assessing changes in the wet weight of mycelial cells. The effect of a sequential combination was examined by pretreating mycelial cells with an antifungal agent, followed by the addition of a second antifungal agent. Pretreatment with amphotericin B (AMPH) followed by miconazole (MCZ) or fluconazole (FLCZ) resulted in better in vitro effects compared with the effect of simultaneous use of the agents. In contrast, pretreatment of mycelial cells with azole antifungal agents, other than MCZ, followed by AMPH, resulted in an increase in the wet weight compared with that recorded after simultaneous incubation with AMPH and azole antifungal agents. Our results showed that combined treatment, of AMPH followed by MCZ or FLCZ, inhibited the growth of A. fumigatus, suggesting that such a regimen may be effective against aspergillosis.