Acute chest syndrome (ACS) is the most serious complication of sickle cell disease. The pathophysiology of ACS may involve lower respiratory tract infection (LRTI), alveolar hypoventilation and atelectasis, bone infarcts-driven fat embolism, and in situ pulmonary artery thrombosis. One of the most challenging issues for the physicians is to diagnose LRTI as the cause of ACS. The use of a respiratory multiplex PCR (mPCR) for the diagnosis of LRTI has not been assessed in sickle-cell adult patients with ACS. To describe the spectrum of infectious aetiologies of severe ACS, using a diagnostic approach combining conventional tests and mPCR. A non-interventional monocenter prospective study involving all the consecutive sickle-cell adult patients with ACS admitted to the intensive care unit (ICU). Microbiological investigation included conventional tests and a nasopharyngeal swab for mPCR. Altogether, 36 patients were enrolled, of whom 30 (83%) had complete microbiological investigations. A bacterial microorganism, mostly Staphylococcus aureus (n = 8), was identified in 11 patients. There was no pneumonia-associated intracellular bacterial pathogen. A respiratory virus was identified in six patients. Using both conventional tests and nasopharyngeal mPCR, a microbiological documentation was obtained in half of adult ACS patients admitted to the ICU. Pyogenic bacteria, especially S. aureus, predominated.