Penicillium costaricense was isolated in air samples from a surgery center in a public hospital in Brazil. The only published data on the occurrence of this species is from caterpillar intestine of Rothschildia lebeau in Costa Rica. The isolate was identified by morphology together with the molecular markers β-tubulin and calmodulin. This report is a warning call to understand the pathogenicity of this species. To the best of our knowledge, this is the first report of P. costaricense in South American and the second in the world.