Differential diagnosis of neurodegenerative parkinsonian syndromes is complicated by overlapping clinical features and frequent co-pathology that challenges the interpretation of single-protein biomarkers. We evaluated a multimodal, minimally invasive biomarker strategy integrating dermal α-synuclein and 4-repeat tau seed amplification assays (SAAs) with serum neurofilament light chain. In a prospective cohort of 166 participants (Parkinson's disease, n = 40; multiple system atrophy, n = 29; progressive supranuclear palsy (PSP), n = 77; healthy controls, n = 20) with independent external validation (63 participants), α-synuclein SAA identified synucleinopathies with high sensitivity but was positive in a subset of PSP, which is consistent with α-synuclein co-pathology. Dermal 4-repeat tau SAA identified PSP with high sensitivity and specificity. Serum neurofilament light chain distinguished multiple system atrophy from Parkinson's disease and correlated with disease severity in PSP. Integrating these complementary biomarkers improved diagnostic discrimination compared with individual markers and enabled further stratification within PSP. These findings support a multimodal biomarker approach for biologically informed diagnosis of parkinsonian syndromes.