This study investigates the effect of nitrogen fertilisation on the anatomical properties of the juvenile wood of the Populus alba L. clone ‘Villafranca’ from an experimental trial near the Drava River in Croatia. Nitrogen was applied for two consecutive years, and the immediate and potential post-treatment effects were investigated. The correlation between annual ring width (ARW) and individual wood anatomical properties was also examined. The fertilisation effect was confirmed after the first year of nitrogen application for all wood anatomical properties except the vessel lumen area (VLA). Fibre length (FL) was reduced, and double cell wall thickness (DCWT), ray area (RA), and cell wall area (CWA) increased. In contrast, the vessel lumen diameter (VLD) and vessel lumen area changed inconsistently between treatments. The second year of nitrogen application was determined to be effective for FL only. Due to the insignificant results in the second year of the application of nitrogen, the post-fertilisation effect of nitrogen fertilisation was not confirmed.