Pythium insidiosum is an oomycete capable of causing a life-threatening disease in humans, called pythiosis. Conventional antifungal drugs are ineffective against P. insidiosum infection. A synthetic mixture of the volatile organic compounds (VOCs) from the endophytic fungus Muscodor crispans strain B23 demonstrates antimicrobial effects against a broad range of human and plant pathogens, including fungi, bacteria, and oomycetes. We studied the in vitro effects of B23 VOCs against 25 human, 1 animal, and 4 environmental isolates of P. insidiosum, compared with a no-drug control. The B23 synthetic mixture, at amounts as low as 2.5 microl, significantly reduced growth of all P. insidiosum isolates by at least 80%. The inhibitory effect of the B23 VOCs was dose-dependent. The growth of all isolates was completely inhibited by a dose of 10.0 microl of B23 VOCs, and all isolates were killed by a dose of 20.0 microl. Synthetic B23 VOCs of M. crispans had inhibitory and lethal effects against all P. insidiosum isolates tested. Further studies are needed to evaluate this mixture for treatment of pythiosis.