Fatigue is one of the most common reasons for failure in existing steel railway bridges, particularly with a riveted structure. Therefore, fatigue assessment is always the most important part of the complex evaluation of existing steel railway bridges. Additionally, a reliable fatigue assessment is often decisive in estimating the remaining service life of a bridge. The purpose of the presented research is to assess the fatigue of a 100-year-old riveted truss railway bridge. The assessment was carried out to decide whether the existing truss superstructure is still suitable for rehabilitation in the context of the ongoing modernisation of the railway line. The fatigue assessment procedure is based on the safe life method in the convention of nominal stresses. The Miner linear cumulative damage theory and the S-N curves according to the relevant European recommendations were also applied. Bridge evaluation revealed that the fatigue life of the steel superstructure was almost exhausted and that its remaining service life is too short to be considered for rehabilitation. It was also confirmed by the presence of fatigue cracks in the deck joints, detected during bridge inspection. Taking into account the assessment results, it was revealed that the existing riveted truss superstructure was not suitable for further use, which was intended to extend the bridge service life by the next 50 years after the modernisation of the railway line.