Recent attempts to establish a molecular time-scale of eukaryote evolution failed to provide a congruent view on the timing of the origin and early diversification of eukaryotes. The major discrepancies in molecular time estimates are related to questions concerning the calibration of the tree. To limit these uncertainties, we used here as a source of calibration points the rich and continuous microfossil record of dinoflagellates, diatoms and coccolithophorids. We calibrated a small-subunit ribosomal RNA tree of eukaryotes with four maximum and 22 minimum time constraints. Using these multiple calibration points in a Bayesian relaxed molecular clock framework, we inferred that the early radiation of eukaryotes occurred near the Mesoproterozoic–Neoproterozoic boundary, about 1100 million years ago. Our results indicate that most Proterozoic fossils of possible eukaryotic origin cannot be confidently assigned to extant lineages and should therefore not be used as calibration points in molecular dating.