Fungal infections cause approximately 1.6 million deaths annually. Diagnosing and treating fungal infections is difficult due to limited access to diagnostic tests and rising antifungal resistance. Extracellular vesicles (EVs) facilitate interactions between fungal cells and hosts, significantly influencing the pathogen-host relationship. Owing to the complexity of fungal EVs and the lack of clinical studies on their roles in human infections, we analysed EVs from serum and urine samples of patients with infections caused by , , and to determine their roles. Using mass spectrometry, we identified sterols, sphingolipids, and fatty acids as key metabolites in the EVs. We quantified cholesterol and ergosterol, confirming the presence of both host and fungal EVs in clinical samples. Our research investigated whether these EVs could modulate the host immune response. We observed a proinflammatory response in murine and human macrophages, characterized by increased cytokines, such as tumour necrosis factor-α, interferon-γ, and interleukin-6, and elevated expression of the inducible nitric oxide synthase gene, a marker of M1 macrophage response. Thus, circulating EVs in patients with fungal infections likely play a role in disease pathophysiology. These findings enhance our understanding of EVs in fungal infections, suggesting potential therapeutic targets for systemic mycoses.