Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a chronic and debilitating disease characterized by unexplained physical fatigue, cognitive and sensory dysfunction, sleeping disturbances, orthostatic intolerance, and gastrointestinal problems. People with ME/CFS often report a prodrome consistent with infections. Using regression, Bayesian and enrichment analyses, we conducted targeted and untargeted metabolomic analysis of plasma from 106 ME/CFS cases and 91 frequency-matched healthy controls. Subjects in the ME/CFS group had significantly decreased levels of plasmalogens and phospholipid ethers (p < 0.001), phosphatidylcholines (p < 0.001) and sphingomyelins (p < 0.001), and elevated levels of dicarboxylic acids (p = 0.013). Using machine learning algorithms, we were able to differentiate ME/CFS or subgroups of ME/CFS from controls with area under the receiver operating characteristic curve (AUC) values up to 0.873. Our findings provide the first metabolomic evidence of peroxisomal dysfunction, and are consistent with dysregulation of lipid remodeling and the tricarboxylic acid cycle. These findings, if validated in other cohorts, could provide new insights into the pathogenesis of ME/CFS and highlight the potential use of the plasma metabolome as a source of biomarkers for the disease.