Airborne Aspergillus spp. are critical pathogens that cause nosocomial infections in hospitals. Despite their importance, little is known about the distribution of Aspergillus species in the indoor air of hospitals in Brazil. We investigated Aspergillus spp. in the indoor air of critical areas in a tertiary hospital in Brazil. Air samples (n = 238) were collected from the intensive care unit (ICU), medical clinic unit (MCU), and urgency and emergency unit (UEU) using an air sampler (100 L/min). Of the 324 Aspergillus isolates, 322 were identified using phenotypic methods, and 37 were identified using DNA sequencing. Aspergillus spp. was grouped into five sections: Fumigati (29.3%), Nidulantes (27.8%), Nigri (27.5%), Flavi (11.7%), and Terrei (3.1%). The predominant species identified via sequencing were Aspergillus sydowii (n = 9), Aspergillus flavus (n = 7), and Aspergilus fumigatus (n = 6). The number of Aspergillus spp. and their sections varied according to the collection day. A. fumigatus was isolated more frequently during winter and in the ICU. This study is the first to demonstrate the diversity of airborne Aspergillus (saprophytic, allergenic, toxigenic, and potentially pathogenic) strains in a hospital located in the Midwest region of Brazil. It contributes to the knowledge of the diversity of cryptic species in the hospital environment.