Autoaggregation of widely dispersed Xylella fastidiosa cells into compact cell masses occurred over a period of hours following 7 to 11 days of growth in microfluidic chambers. Studies involving the use of mutants defective in polarly positioned type I (fimA-negative), type IV (pilB-negative), or both type I and IV (fimA- and pilO-negative) pili revealed the importance and role of pili in the autoaggregation process.