Abstract Lipid and cholinergic mediators are inflammatory regulators, but their role in the immunopathology of COVID-19 is still unclear. Here, we used human blood and tracheal aspirate (TA) to investigate whether acetylcholine (Ach), fatty acids (FAs), and their derived lipid mediators (LMs) are associated with COVID-19 severity. First, we analyzed the perturbation profile induced by SARS-CoV-2 infection in the transcriptional profile of genes related to the ACh and FA/LM pathways. Blood and TA were used for metabolomic and lipidomic analyses and for quantification of leukocytes, cytokines, and ACh. Differential expression and coexpression gene network data revealed a unique transcriptional profile associated with ACh and FA/LM production, release, and cellular signaling. Transcriptomic data were corroborated by laboratory findings: SARS-CoV-2 infection increased plasma and TA levels of arachidonic acid, 5-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid, 11-hydroxy-5Z,8Z,12E,14Z-eicosatetraenoic acid, and ACh. TA samples also exhibited high levels of PGE2, thromboxane B2, 12-oxo-5Z,8Z,10E,14Z-eicosatetraenoic acid, and 6-trans-leukotriene B4. Bioinformatics and experimental approaches demonstrated robust correlation between transcriptional profile in Ach and FA/LM pathways and parameters of severe COVID-19. As expected, the increased neutrophil-to-lymphocyte ratio, neutrophil counts, and cytokine levels (IL-6, IL-10, IL-1β, and IL-8) correlated with worse clinical scores. Glucocorticoids protected severe and critical patients and correlated with reduced Ach levels in plasma and TA samples. We demonstrated that pulmonary and systemic hyperinflammation in severe COVID-19 are associated with high levels of Ach and FA/LM. Glucocorticoids favored the survival of patients with severe/critical disease, and this effect was associated with a reduction in ACh levels.