In 2018, during the construction of a motorway in the East Bohemian Region near the town of Ostrov (Czech Republic), archaeologists excavated a structure of a wooden water well lining with a square base area of 80 × 80 cm and 140 cm in height. Due to the excellent conservation of the oak timbers, studies of technological details and precise tree-ring dating were possible. The used trees were felled in the years 5256/55 BC, which makes this well the oldest dendrochronologically dated archaeological wooden construction worldwide. It is the third well from the Early Neolithic period that has been discovered in the Czech Republic within the last four years. The design consists of grooved corner posts with inserted planks. This type of construction reveals advanced technical know-how and, till now, is the only known type from this region and time period. Thanks to the combination of annually resolved and absolutely dated tree-ring widths (TRWs), the Czech oak TRW chronology has been significantly extended back to 5481 BC. Wood anatomical identification of fragments from the well filling show mainly oak (Quercus spp.) and hazel (Corylus spp.), indicating a local forest composition dominated by these taxa. The shape of the individual structural elements and tool marks preserved on their surface confirm sophisticated carpentry skills. Based on these observations, we established a model for the “chaîne opératoire” from forest utilization to the final artefact at the beginning of the Early Neolithic period.