Blastocystis is a commonly occurring gut microorganism that is present in one to two billion people worldwide. Despite being one of the most prevalent eukaryotic gut microorganisms found in the human gastrointestinal tract, its biology remains elusive and its role in the microbiota is not fully understood yet. Much of the lack of understood is due to the challenge of culturing Blastocystis in isolation (axenically) in vitro. Nine xenic subtypes of Blastocystis were cultured in vitro, and their bacterial composition, and metabolite profiles were analysed using 16S amplicon sequencing and 1H-NMR metabolomics. At phylum level all subtypes are dominated by Proteobacteria, Bacteroidetes and Firmicutes, but even at this broad taxonomic level there are clear differences between subtypes. It was seen that ST3 exists alongside a distinct prokaryotic profile and that as a result, the metabolites present in the culture are also distinct from the other subtypes investigated. ST3 was seen to be enriched in SCFAs and amino acid derivatives, whilst having a lower relative abundance of benzoate. Methanobrevibacter and Enterobacter were seen to be potential biomarkers of ST3. Further investigation into ST3 combined microbiome, metabolomics and transcriptomics data to obtain a more detailed picture of the biology of this ST. It is evident that there are subtype-specific microenvironments of Blastocystis in culture, and these findings highlights the role that non-prokaryotic microorganisms could be having in the context of the microbiome.