This report presents 50 AMS radiocarbon dates from wood charcoal samples extracted from newly uncovered Neolithic settlement layers at the archaeological site of Amzabegovo (Sveti Nikole, North Macedonia). It constitutes the largest radiocarbon dataset from a Neolithic site in North Macedonia. The calibrated dates span 6391–5635 cal BC (95.4%), falling within the Early and Middle Neolithic. With an inferred occupation onset potentially as early as 6391–6236 cal BC (95.4%), Amzabegovo provides the earliest securely dated Neolithic settlement layers in the Ovče Pole catchment area and serves as an important chronological reference point for Early Neolithic expansion in the Vardar River basin. The consideration of the radiocarbon dates from the American excavations led by Marija Gimbutas in 1969–1970 highlights both the possibilities and the limitations of juxtaposing the old and new datasets. Future analyses of the archaeological stratigraphy and material from Amzabegovo hold the potential to offer insights into the Neolithic period in the southern Balkans and beyond, with the new radiocarbon dates significantly enhancing our understanding of the region’s chronological developments.