The majority of bridges and quay walls in the centre of Amsterdam are supported by 100–300 years-old wooden foundation piles subjected to bacterial decay. 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. In this study, micro-drilling measurements were employed to capture the amount of decay and remaining short-term compressive strength of the historic wooden piles. The applicability of micro-drilling was studied on 60 wooden piles with various decay levels, retrieved after 100–295 years of service life. An algorithm was developed for analysing the micro-drilling signals, aimed at determining the decayed outer layer of the piles’ cross section, and validated with the results of mechanical testing on the piles. The micro-drilling technique is now used on a large scale in Amsterdam, supporting the assessment of the wooden foundation piles in the city.