Updated project metadata. Background: Congenital cytomegalovirus (cCMV) is a common intrauterine infection, leading to neurodevelopmental disabilities. Early prognostic assessment of the severity of cCMV has remained an ongoing challenge. We aimed to identify amniotic fluid biomarkers of the severity of cCMV-related fetal brain injury. Methods: Global proteome analysis was performed in mid-gestation amniotic fluid samples, comparing fetuses with severe cCMV to asymptomatic CMV-infected fetuses (the discovery cohort). The levels of selected differentially-excreted proteins were further determined by specific immunoassays, and evaluated in an independent validation cohort, including clinically-blinded amniotic fluid of CMV-infected fetuses from unrelated centers. Findings: Proteome analysis identified 29 amniotic fluid proteins with distinct abundance in fetuses with severe-, compared to asymptomatic, cCMV. Pathway analysis of the differentially-excreted proteins revealed enriched pathways of inflammatory response, cellular compromise, immunological disease, organismal injury, and neurological disease, underlining a link between excessive inflammation at the maternal-fetal interface and the development of cCMV-related fetal brain damage. Importantly, the levels of two of these proteins, chemerin and galectin-3-binding protein (Gal-3BP), selected for validation, demonstrated 88.2% sensitivity (each), 100-96.2% specificity, 100-93.8% positive predictive value, and 92.9-92.6% negative predictive value, with 0.98-0.97 area under the curve (for chemerin - Gal-3BP, respectively) in differentiating 17 fetuses with severe cCMV from 26 asymptomatic CMV-infected fetuses. Interpretation: These results identify the immunomodulatory proteins chemerin and Gal-3BP as new highly predictive amniotic fluid biomarkers of cCMV severity, which could guide early prognostic stratification and potential personalized treatment of cCMV-infected fetuses. Funding: Israel Science Foundation; Research Fund of the Hadassah Medical Organization