The majority of the bridges in the historic city centre of Amsterdam are supported by wooden foundation piles. Most of these were constructed 100–300 years ago, currently raising concerns about potential safety issues. The wooden piles under the bridges remain entirely under the water table, potentially subjected to bacterial decay in anaerobic conditions. Bacterial degradation proceeds at a slow rate, allowing the piles to perform their function for many years, although causing a reduction of their load-carrying capacity over time. To this end, a large experimental campaign was conducted to characterize the material and mechanical properties in relation to biological decay of 60 spruce and fir piles, dated back to 1727, 1886 and 1922, retrieved from two bridges in Amsterdam. Large-scale compression tests were carried out on 201 pile-segments extracted from head, middle-part and tip of the piles to determine their remaining short-term compressive strength. Micro-drilling measurements were conducted on each pile and analysed with a TU-Delft-developed algorithm, aimed at determining the soft shell – the width of the decayed outer layer of the piles’ cross section. Micro-drilling allowed to accurately assess the remaining sound cross section of the pile, which resulted to be well correlated to its mechanical properties. The extent of decay throughout the cross-section of the piles was assessed within sapwood and heartwood through experimental models from literature and validated with Computed Tomography (CT) scanning. This allowed to identify that bacterial decay was only present in the non-durable sapwood, even in very degraded piles. Moreover, the soft shell resulted to be rather uniform along the piles’ length. The analysis of decay, supported by micro-drilling and CT scans, allowed to develop experimental equations to predict the remaining short-term compressive strength along the pile length. The micro-drilling technique is now used on a large scale in Amsterdam, supporting the assessment of the remaining load carrying-capacity of wooden foundation piles in the city, aiding in the planning of conservation, maintenance and preservation strategies.