The Conception and St. John’s Groups of southeastern Newfoundland contain some of the oldest known fossils of the Ediacaran macrobiota. The Mistaken Point Ecological Reserve UNESCO World Heritage Site is an internationally recognized locality for such fossils and hosts early evidence for both total group metazoan body fossils and metazoan-style locomotion. The Mistaken Point Ecological Reserve sedimentary succession includes ∼1500 m of fossil-bearing strata containing numerous dateable volcanogenic horizons, and therefore offers a crucial window into the rise and diversification of early animals. Here we present six stratigraphically coherent radioisotopic ages derived from zircons from volcanic tuffites of the Conception and St. John’s Groups at Mistaken Point Ecological Reserve. The oldest architecturally complex macrofossils, from the upper Drook Formation, have an age of 574.17 ± 0.66 Ma (including tracer calibration and decay constant uncertainties). The youngest rangeomorph fossils from Mistaken Point Ecological Reserve, in the Fermeuse Formation, have a maximum age of 564.13 ± 0.65 Ma. Fossils of the famous “E” Surface are confirmed to be 565.00 ± 0.64 Ma, while exceptionally preserved specimens on the “Brasier” Surface in the Briscal Formation are dated at 567.63 ± 0.66 Ma. We use our new ages to construct an age-depth model for the sedimentary succession, constrain sedimentary accumulation rates, and convert stratigraphic fossil ranges into the time domain to facilitate integration with time-calibrated data from other successions. Combining this age model with compiled stratigraphic ranges for all named macrofossils within the Mistaken Point Ecological Reserve succession, spanning 76 discrete fossil-bearing horizons, enables recognition and interrogation of potential evolutionary signals. Peak taxonomic diversity is recognized within the Mistaken Point and Trepassey Formations, and uniterminal rangeomorphs with undisplayed branching architecture appear several million years before multiterminal, displayed forms. Together, our combined stratigraphic, paleontological, and geochronological approach offers a holistic, time-calibrated record of evolution during the mid–late Ediacaran Period and a framework within which to consider other geochemical, environmental, and evolutionary data sets.