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Three years ago, a novel association between myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and the murine retrovirus XMRV was published.[1] Since then, 191 papers have been published on the subject (NCBI PubMed, accessed 6 November 2012), largely disproving the initial association, a trend confirmed by a recent multicentre blinded trial which definitively concluded that there is no association between ME/CFS and XMRV.[2] It is therefore time to revisit the investigation of ME/CFS aetiology. Metagenomics offers a promising new opportunity for hypothesis discovery in microbial associations with ME/CFS, and we describe herein the technical basis of this approach and its advantages in aetiological agent investigation.