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Background: The microbiome of the ocular surface has been characterised, but only limited in-formation is available on a possible silent intraocular microbial colonisation in normal eyes. Therefore, we performed next generation sequencing (NGS) of 16S rDNA genes in aqueous hu-mor. Methods: Aqueous humor was sampled from three patients during cataract surgery. Air swabs, patient’s and healthy donor conjunctival swab served as controls. Following DNA extraction, the V3 and V4 hypervariable regions of the 16S rDNA gene were amplified and sequenced fol-lowed by denoising. The resulting Amplicon Sequence Variants were matched to a subset of the Ribosomal Database Project 16S database. The deduced bacterial community was then statis-tically analysed. Results: DNA content in all samples was low (0-1.49 ng/µl) but sufficient for analysis. Main phyla in all samples were Acinetobacteria (48%), Proteobacteria (26%), Firmicutes (14%), Acidobac-teria (8%) and Bacteroidetes (2%). Patients conjunctival control samples and anterior chamber fluid showed similar patterns of bacterial species containing many waterborne species. Non-disinfected samples showed a different bacterial spectrum than the air swab sample. Conclusions: The data confirm the existence of an ocular surface microbiome. A distinct intraoc-ular microbiome was not discernable from the background, suggesting the absence of an intra-ocular microbiome in normal eyes.