This study evaluated the in vitro activity of aminoglycoside antibiotics and tigecycline against Pythium insidiosum. The susceptibility tests were carried out using the broth microdilution method in accordance with the CLSI document M38-A2. MIC values for gentamicin, neomycin, paromomycin, and streptomycin ranged from 32 to 64 mg/liter, and the minimal fungicidal concentration (MFC) ranged from 32 to 128 mg/liter, which are incompatible with safe concentrations of these drugs in plasma in vivo. Tigecycline showed the lowest MIC (0.25 to 2 mg/liter) and MFC (1 to 8 mg/liter) range values. The in vitro susceptibility observed to tigecycline makes this drug a good option in future tests in vitro and in vivo for the management of pythiosis.