Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating multisystemic disease of unknown etiology, affecting thousands of individuals worldwide. Its diagnosis still relies on ruling out medical problems leading to unexplained fatigue due to a complete lack of disease-specific biomarkers. Our group and others have explored the potential value of microRNA profiles (miRNomes) as diagnostic tools for this disease. However, heterogeneity of participants, low numbers, the variety of samples assayed, and other pre-analytical variables, have hampered the identification of disease-associated miRNomes. In this study, our team has evaluated, for the first time, ME/CFS miRNomes in peripheral blood mononuclear cells (PBMCs) and extracellular vesicles (EVs) from severely ill patients recruited at the monographic UK ME biobank to assess, using standard operating procedures (SOPs), blood fractions with optimal diagnostic power for a rapid translation of a miR-based diagnostic method into the clinic. Our results show that routine creatine kinase (CK) blood values, plasma EVs physical characteristics (including counts, size and zeta-potential), and a limited number of differentially expressed PBMC and EV miRNAs appear significantly associated with severe ME/CFS (p < 0.05). Gene enrichment analysis points to epigenetic and neuroimmune dysregulated pathways, in agreement with previous reports. Population validation by a cost-effective approach limited to these few potentially discriminating variables is granted.