Aspergillosis remains a major cause of mortality in captive and rehabilitated seabirds. To date, there has been poor documentation of fungal (particularly Aspergillus spp.) burdens in natural seabird loafing and roosting sites compared with fungal numbers in rehabilitation or captive settings and the various microenvironments that seabirds are exposed to during the rehabilitation process. This study compares fungal, particularly Aspergillus spp., burdens potentially encountered by seabirds in natural and rehabilitation environments. Differences among the various microenvironments in the rehabilitation facility were evaluated to determine the risk of infection when seabirds are experiencing high stress and poor immune function. Aspergillus spp. counts were quantified in three wildlife rehabilitation centers and five natural seabird loafing and roosting sites in northern California using a handheld impact air sampler and a water filtration system. Wildlife rehabilitation centers demonstrated an increase in numbers of conidia of Aspergillus spp. and Aspergillus fumigatus in air and water samples from select aquatic bird rehabilitation centers compared with natural seabird environments in northern California. Various microenvironments in the rehabilitation facility were identified as having higher numbers of conidia of Aspergillus spp. These results suggest that periodic monitoring of multiple local areas, where the birds spend time in a rehabilitation facility, should be done to identify “high risk” sites, where birds should spend minimal time, or sites that should be cleaned more frequently or have improved air flow to reduce exposure to fungal conidia. Overall, these results suggest that seabirds may be more likely to encounter Aspergillus spp. in various microenvironments in captivity, compared with their native habitats, which could increase their risk of developing disease when in a debilitated state.