The discovery of various strains belonging to three subspecies of Xylella fastidiosa in Europe has raised concerns about the potential spread of the bacteria to Northern regions. It is crucial to evaluate the susceptibility of European flora, which has not been exposed to this pathogen before. In a biosafety facility, we mechanically inoculated KLN59.3 GFP-labelled X. fastidiosa at 22°C and 28°C to assess the susceptibility of Salicaceae, including Populus tremula, Populus canescens, Salix alba, and Salix caprea. The movement and multiplication of bacteria in plants were examined using PCR, real-time PCR, confocal or scanning electron microscopy. After nine months, all plants tested positive for X. fastidiosa, except for 57% of P. canescens under the 22°C growing conditions. Bacteria were detected up to 120 cm from the inoculation point for S. alba and were found in the roots of all species. Successful isolation was achieved for S. alba and P. tremula. The average CFU/g of plant tissue per species ranged from 1.5E+03 to 3.5E+06, with the highest figures for P. tremula, which also showed a high number of totally obstructed vessels observed by confocal microscopy. Additionally, more than 400 endophytic bacterial isolates obtained from Salicaceae xylem sap, extracted using a Scholander pressure chamber, are currently under investigation as potential antagonist agents.