The biological characteristics of Xylella fastidiosa (Xf) were investigated in vitro by examining the effect of pure xylem fluid from Pierce's disease (PD)-resistant Vitis smalliana grapevines on Xf cell multiplication, aggregation, biofilm formation, and motility in microfluidic flow chambers. The aggregations of large clumps were formed in artificial medium, whereas small clumps were observed in V. smalliana xylem fluid. Xylem fluid from V. smalliana negatively affected twitching motility and repressed biofilm formation of Xf, whereas artificial medium enhanced twitching motility and supported robust biofilm development. The results indicate that chemical components of V. smalliana xylem fluid may affect the function of type IV pili of Xf, leading to reduced twitching motility and aggregation as compared to artificial medium.