Invasive mycosis has significantly increased in frequency among immunocompromised hosts leading to excessive morbidity and mortality. The combination of sulfamethoxazole (SMX) and trimethoprim (TMP) has been used extensively for the treatment and prophylaxis of infections by various microbes. The purpose of this study is to estimate the anti-fungal activity of SMX-TMP and examine the mechanism of activity. To investigate the antimicrobial activity of SMX-TMP in vitro, a mixture of SMX and TMP at 5 : 1 was serially diluted and added to potato dextrose agar medium or C-limiting agar medium. Aspergillus species were inoculated on the medium plate with SMX-TMP. The growth of A. fumigatus and A. oryzae was inhibited by addition of SMX-TMP. The anti-Aspergillus effect depended on not TMP but SMX and that was inhibited by p-aminobenzoic acid (PABA). A. niger was not sensitive against SMX-TMP in PDA medium, but sensitive in C-limiting medium. Those results showed that the activity depends on culture medium. Furthermore, addition of human serum did not influence the activity of SMX. The finding in this study suggested that SMX might be effective against Aspergillus species in clinical practice and prophylaxis treatment.