Xanthomonasarboricola pv. pruni (Xap) causes bacterial spot of stone fruit and almond, an important plant disease with a high economic impact. Biofilm formation is one of the mechanisms that microbial communities use to adapt to environmental changes and to survive and colonize plants. Herein, biofilm formation by Xap was analyzed on abiotic and biotic surfaces using different microscopy techniques which allowed characterization of the different biofilm stages compared to the planktonic condition. All Xap strains assayed were able to form real biofilms creating organized structures comprised by viable cells. Xap in biofilms differentiated from free-living bacteria forming complex matrix-encased multicellular structures which become surrounded by a network of extracellular polymeric substances (EPS). Moreover, nutrient content of the environment and bacterial growth have been shown as key factors for biofilm formation and its development. Besides, this is the first work where different cell structures involved in bacterial attachment and aggregation have been identified during Xap biofilm progression. Our findings provide insights regarding different aspects of the biofilm formation of Xap which improve our understanding of the bacterial infection process occurred in Prunus spp and that may help in future disease control approaches.