We aimed to assess the prognostic value of serum β-synuclein (β-syn), neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP) in patients with moderate-to-severe acute ischemic stroke. We measured β-syn, GFAP and NfL in serum samples collected one day after admission in 30 adult patients with moderate-to-severe ischemic stroke due to middle cerebral artery (MCA) occlusion. We tested the associations between biomarker levels and clinical and radiological scores (National Institute of Health Stroke Scale scores, NIHSS, and Alberta Stroke Program Early CT Score, ASPECTS), as well as measures of functional outcome (modified Rankin Scale, mRS). Serum biomarkers were significantly associated with ASPECTS values (β-syn p = 0.0011, GFAP p = 0.0002) but not with NIHSS scores at admission. Patients who received mechanical thrombectomy and intravenous thrombolysis showed lower β-syn (p = 0.029) und NfL concentrations (p = 0.0024) compared to patients who received only mechanical thrombectomy. According to median biomarker levels, patients with high β-syn, NfL or GFAP levels showed, after therapy, lower clinical improvement (i.e., lower 24-h NIHSS change), higher NIHSS scores during hospitalization and higher mRS scores at 3-month follow-up. Elevated serum concentrations of β-syn (p = 0.016), NfL (p = 0.020) or GFAP (p = 0.010) were significantly associated with 3-month mRS of 3–6 vs. 0–2 even after accounting for age, sex and renal function. In patients with moderate-to-severe acute ischemic stroke, serum β-syn, NfL and GFAP levels associated with clinical and radiological scores at different timepoints and were able to predict short- and middle-term clinical outcomes.