Although the brain has always been viewed as a sterile organ, recent studies have suggested the existence of a ‘brain microbiome,’ perturbations of which could cause neuroinflammatory conditions. Unfortunately, experiments aimed at detecting a brain microbiome are limited by a low bacterial biomass. Bacteria must be detected through an overwhelming amount of host DNA, and the low biomass additionally raises the risk of amplifying exogenous contaminants. A recent study tested the hypothesis that there is a bacterial brain microbiome. Using 16S rRNA sequencing, researchers evaluated brain samples from healthy individuals and individuals suffering from Parkinson’s disease (PD), along with murine brains. They found that while amplicon sequencing detected bacterial signals in both human and murine brains, the estimated bacterial biomass was extremely low. Careful reanalysis suggested that bacterial signals were explained by exogenous DNA contamination and false-positive amplification of host DNA. Additionally, all tested bioinformatic pipelines mis-classified some amplified host DNA sequences as bacterial 16S rDNA, leading to false positives during analysis. While further study is needed, these results do not support the existence of a brain microbiome or bacterial infection in the brains of individuals with PD, highlighting the need for stringent negative and positive controls for both laboratory experiments and bioinformatic pipelines.