BACKGROUND. Cytomegalovirus (CMV) is the most common intrauterine infection, leading to infant brain damage. Prognostic assessment of CMV-infected fetuses has remained an ongoing challenge in prenatal care, in the absence of established prenatal biomarkers of congenital CMV (cCMV) infection severity. We aimed to identify prognostic biomarkers of cCMV-related fetal brain injury. METHODS. We performed global proteome analysis of mid-gestation amniotic fluid samples, comparing amniotic fluid of fetuses with severe cCMV with that of asymptomatic CMV-infected fetuses. The levels of selected differentially excreted proteins were further determined by specific immunoassays. RESULTS. Using unbiased proteome analysis in a discovery cohort, we identified amniotic fluid proteins related to inflammation and neurological disease pathways, which demonstrated distinct abundance in fetuses with severe cCMV. Amniotic fluid levels of 2 of these proteins — the immunomodulatory proteins retinoic acid receptor responder 2 (chemerin) and galectin-3–binding protein (Gal-3BP) — were highly predictive of the severity of cCMV in an independent validation cohort, differentiating between fetuses with severe (n = 17) and asymptomatic (n = 26) cCMV, with 100%–93.8% positive predictive value, and 92.9%–92.6% negative predictive value (for chemerin and Gal-3BP, respectively). CONCLUSION. Analysis of chemerin and Gal-3BP levels in mid-gestation amniotic fluids could be used in the clinical setting to profoundly improve the prognostic assessment of CMV-infected fetuses.