The majority of bridges in Amsterdam’s historic city centre are founded on timber foundation piles, typically 12 to 15 meters in length, which function as end-bearing elements reaching the underlying stiff sand layer. Currently, many timber foundations have been in service for up to 300 years, raising concerns about their remaining load-bearing capacity and the overall safety of the bridges they support. The timber piles beneath the bridges remain fully submerged, where their outer cross-section is exposed to slow bacterial decay in anaerobic conditions, leading to a reduction in the sound load-bearing core of the piles. Despite decay, the piles maintain their structural capacity for many years, although their load-bearing capacity gradually decreases over time. The two primary risks are attributed to the applied loads exceeding the load-bearing capacity of the timber piles, and progressive damage accumulation over time due to sustained loading, ultimately leading to large settlements or failure. In this context, 201 pile segments were extracted from two bridges in Amsterdam and mechanically characterised, with respect to their amount of biological decay and service durations ranging from 100 to 300 years. Large-scale compression tests were carried out to determine the remaining saturated short-term compressive strength of the piles. Micro-drilling measurements were conducted to assess the amount of bacterial decay, validated with Computed Tomography (CT) scanning. Finally, this study investigated the load history and current loads acting on the timber foundation of bridge 30 (De Isa van Eeghenbrug) in Amsterdam, to assess the remaining load-bearing capacity of the piles, considering mechanical damage and decay.