In the historic city centre of Amsterdam (NL), the predominant foundation system is comprised of wooden piles. Due to their placement below the water table, these foundations are susceptible to bacterial decay. This study aims to investigate and compare various methods for characterizing decay patterns within the cross sections of piles retrieved from two bridges in Amsterdam. The examined piles span different construction years: three originate from 1727, four from 1886, and two from 1922. Following extraction, the piles were transported to TU Delft Stevin II Laboratory, where they underwent further subdivision into three segments, each representing the head, middle, and tip, resulting in a total of 27 segments. The effects of bacterial decay were characterised by performing micro-drilling measurements, small-scale material and compressive tests on prismatic samples extracted from the segments' cross sections, computed tomography scans, and light microscopy observations. Microscopic examination revealed severe degradation in all segments dating back to 1727, extending 20–50 mm from their surface. This outcome was also confirmed by the other adopted methods: the corresponding prisms had large moisture contents and poor mechanical properties, while low basic densities and drilling amplitudes were obtained from CT scans and micro-drilling measurements, respectively. On the contrary, the internal sections of the 1727 segments exhibited no evidence of decay and demonstrated properties consistent with those observed in sound segments from 1886 and 1922. Finally, the observed gradients of density, strength, and stiffness were well correlated with micro-drilling measurements, which can therefore be reliably used as on-site assessment method to reconstruct the properties of the piles.