The quantification of α-synuclein (α-syn) aggregates has emerged as a promising biomarker for synucleinopathies. Assays that amplify and detect such aggregates have revealed the presence of seeding-competent species in biosamples of patients diagnosed with Parkinson’s disease. Multiple species such as oligomers and amyloid fibrils, are formed during the aggregation of α-synuclein and these species are likely to co-exist in biological samples. These discrete species are difficult to quantify in ensemble assays and thus remains unclear which species(s) are contributing to the signal detected in amplification assays. To identify which species are detected in seeding assays, recombinant oligomers and preformed fibrils were produced and purified to characterise their individual biochemical and seeding potential. Here, we used single molecule spectroscopy to track the formation and purification of oligomers and fibrils reconstituted from recombinant α-syn monomers at the single particle level and compare their respective seeding potential in an amplification assay. We demonstrate that oligomers can be completely isolated from fibrils by coupling single molecule detection and size-exclusion chromatography. Our results reveal that a small fraction of oligomers was indeed seeding-competent yet had a different seeding signature compared to preformed fibrils. Our data suggest that even a low number of preformed fibrils present in biosamples are likely to dominate the response in amplification assays and the quantitative technique developed herein may be applicable to study seeding properties of other prion-like proteins.