The possibility that patients diagnosed with Alzheimer's disease (AD) have disseminated fungal infection has been recently advanced by the demonstration of fungal proteins and DNA in nervous tissue from AD patients. In the present study, next-generation sequencing (NGS) was used to identify fungal species present in the central nervous system (CNS) of AD patients. Initially, DNA was extracted from frozen tissue from four different CNS regions of one AD patient and the fungi in each region were identified by NGS. Notably, whereas a great variety of species were identified using the Illumina platform, Botrytis cinerea and Cryptococcus curvatus were common to all four CNS regions analyzed. Further analysis of entorhinal/cortex hippocampus samples from an additional eight AD patients revealed a variety of fungal species, although some were more prominent than others. Five genera were common to all nine patients: Alternaria, Botrytis, Candida, Cladosporium, and Malassezia. These observations could be used to guide targeted antifungal therapy for AD patients. Moreover, the differences found between the fungal species in each patient may constitute a basis to understand the evolution and severity of clinical symptoms in AD.