Background: Upregulation of PD-L1 (B7-H1, CD274) on eosinophils (Eos) discriminates moderate/severe (MOD) and mild forms of COVID-19. PD-L1 is a ligand of PD-1 (CD279), a coinhibitory receptor (immune checkpoint) expressed by T cells and thus a gatekeeper for broad adaptive immune suppression. Outside COVID-19, PD-L1/PD-1-mediated synapses enable Eos to downregulate T cell responses, mainly in the context of Eos activation and degranulation. We sought to determine Eos activation during COVID-19. Method: Eosinophil-derived neurotoxin (EDN), a granular RNase specific of Eos, was measured with an experimental fluoroimmunoenzyme assay in the serum of 79 COVID-19 patients (PCR-confirmed) at 0–180 days post-symptom onset (PSO) and of 15 PCR-negative controls. Serum (s) EDN, Eos blood counts and the EDN/blood Eos ratio were analyzed as a function of COVID-19 severity and time PSO. Results are expressed as median and interquartile range (IQR). Results: Patients experienced mild (60) or MOD (19) COVID-19. The former received outpatient care. Moderate (MOD) required hospitalization and oxygen but not intensive care. MOD (59.41–71) were older than controls (32.24–54) but not mild (39.26–51). Overall, sEDN levels in mild COVID-19 at any time PSO were lower than in controls or MOD: 49 (25–75), 69 (36–98) and 78 (54–125) µg/L, p < 0.05. sEDN decreased in mild COVID-19 as early as the first 24h PSO, remained significantly lower until day 22, and was still lower, albeit not significantly, after 6 months. Eos counts dropped in MOD (median<0.005 giga/L vs mild 0.06, controls 0.14, p < 0.05). Patient EDN/Eos ratio increased during the first 24h PSO, peaked during the 1st week, then decreased progressively. Peak levels of the EDN/Eos ratio, attained during the first week, were substantially higher in MOD (27325, 15800–38850 vs mild 864, 511–2027 and control 543, 420–1560, p < 0.001). EDN/Eos ratio values returned to baseline at day 22 PSO in mild forms, but only at 6 months in MOD. Conclusion: Analysis of sEDN/Eos ratio levels and kinetics suggests COVID-19-related eosinopenia and tissue emigration associate with early Eos activation, proportional to severity and peaking during the first week PSO. Early Eos activation might contribute to downstream modulation of adaptive immune responses, notably T cells, and COVID-19-related immune exhaustion. Moreover, released EDN may contribute to chemotaxis and local and systemic inflammation, hallmarks of COVID-19 severity.