Objective To investigate the in vitro antifungal activity of domestic amphotericin B and other common antifungal agents against the clinical isolates associated with invasive fungal disease. Methods Minimal inhibitory concentration (MIC) or minimum effect concentration (MEC) of domestic amphotericin B and other common antifungal agents against 108 strains associated with invasive fungal disease were tested by using CLSI (Clinical and Laboratory Standards Institute) broth microdilution method M27-A3 and M38-A2. Results The MICs of amphotericin B against almost all strains tested were less than or equal to 2 mg/L, except Aspergillus terreus, Scedosporium prolificans and S. apiospermum (MIC range of 2-4 mg/L, >16 mg/L, 1-8 mg/L, respectively). The MICs of amphotericin B against azole-resistant Candida and Aspergillus were less than or equal to 2 mg/L. The MICs of fluconazole against all the filamentous fungi tested were greater than 64 mg/L. The MICs of itraconazole against Fusarium solani, S. prolificans and Mucor circinelloides were 8 mg/L, >16 mg/L, >16 mg/L, respectively. The MICs of voriconazole against F. solani, S. prolificans and all Mucor tested were greater than 16 mg/L. The MICs of posaconazole against Fusarium solani, S. prolificans and Mucor circinelloides were all greater than 16 mg/L. The MICs/MECs of caspofungin and micafungin against Cryptococcus, Trichosporon, Mucor, F. solani and Scedosporium were all greater than 16 mg/L. The MECs of caspofungin and micafungin against S. prolificans were 8 and 4 mg/L, respectively. Conclusion Compared with other common antifungal agents, amphotericin B is active against the clinical isolates associated with invasive fungal disease, except A. terreus and Scedosporium.