<p>The misdiagnosis of acute febrile illness (AFI) aetiology is associated with antibiotic overuse and improper patient management. To develop novel strategies, a more comprehensive understanding of the pathophysiological mechanisms underlying the early response to different causes of fever is essential.</p><p>Here we examined the host response to AFI, through the retrospective investigation of serum samples from febrile patients with bacterial infection (<i>n</i> = 52) or malaria (<i>n</i> = 73). The systemic levels of CXCL10, IFNγ, IL-2, IL-4, IL-6, IL-8, procalcitonin (PCT), TRAIL, ICAM-1, VCAM-1, osteopontin, TNFα, MMP-2 and MMP-9 were measured by multiplex immunoassays, while the relative abundance of 37 different surface markers of circulating extracellular vesicles (EVs) was determined using the MACSplex kit (Miltenyi Biotec).</p><p>Markers of endothelial activation (CD29⁺, CD62P⁺, CD9⁺ EVs) and immune cell migration (MMP-2 and MMP-9) appeared elevated in bacterial AFI (q-value &lt; 0.05). Mediators associated with antigen recognition (CD8⁺, CD81⁺, HLA-DR/DP/DQ⁺ EVs) and cytokine storm (IFNγ, CXCL10, TNFα) were instead prominent in malaria infection (q-value &lt; 0.005). Interestingly, HLA-DR/DP/DQ⁺ EVs distinguished between bacterial and malaria AFI with area under the ROC curve (AUC) of 0.87, not significantly different from platelets’ AUC (0.92), which was the best individual discriminator.</p><p>Our results showed different biological processes as associated with bacterial and malaria AFI, paving the way for the identification of novel potential markers for a timely and reliable AFI diagnosis. The study also provides novel insights on the potential role of EVs as mediators of pro-inflammatory signalling during acute fever, which should be investigated more comprehensively in vitro.</p>