Aspergillosis continues to be one of the most important causes of disease in captive penguins. As such, designing exhibits and holding areas that minimize the risk of aspergillosis is of great interest; however, very little has been published regarding this topic. The goal of this study was to assess total fungal spore loads as well as the loads of Aspergillus spp. encountered in multiple indoor and outdoor microenvironments around the exhibit for a large colony of African penguins (Spheniscus demersus). Air samples were collected via impaction at the microenvironments on a monthly basis over a 1-yr period. Results of this study indicated seasonal trends in both total fungal spore loads as well as Aspergillus spp. loads, with the lowest levels encountered during January through April. During the warmer, more humid spring, summer, and fall months when outdoor microenvironments experienced the highest fungal loads, the air-handling system and the pleated filters used indoors are thought to have reduced the fungal loads in the indoor microenvironments compared with the outdoor microenvironments. Additionally, surrounding planting beds were thought to contribute to the higher total fungal loads and Aspergillus spp. loads in the outdoor microenvironments. Results of this study are useful in understanding the factors that contribute to Aspergillus spp. loads in areas that house penguins, and can be used in guiding design, construction, and landscaping of penguin enclosures.