A growing body of literature suggests that alterations in the human microbiome are causative of disease initiation and progression. Aykut et al. present data supporting the argument that alterations in the gut fungal microbiome (the “mycobiome”), along with the presence of fungal elements within pancreatic tissue (specifically those of the genus Malassezia), are associated with pancreatic oncogenesis. Upon analyzing the human sequencing data presented in the original manuscript, we found few fungal reads in pancreatic tissue samples and did not identify differences in pancreatic or gut mycobiome composition between healthy and pancreatic ductal adenocarcinoma (PDAC) patients. Our re-analysis of these data does not support an association between an intrinsic pancreatic mycobiome and the development of human PDAC, and illustrates the challenges in analyzing microbiome sequencing data from low biomass samples.