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The bacterial wilt pathogenRalstonia pseudosolanacearum (Rps)colonizes plant xylem vessels and blocks the flow of xylem sap by its biofilm (comprising of bacterial cells and extracellular material), resulting in devastating wilt disease across many economically important host plants including tomatoes. The technical challenges of imaging the xylem environment, along with the use of artificial cell culture plates and media in existingin vitrosystems, limit the understanding ofRpsbiofilm formation and its infection dynamics. In this study, we designed and built a microfluidic system that mimicked the physical and chemical conditions of the tomato xylem vessels, and allowed us to dissectRpsresponses to different xylem-like conditions. The system, incorporating functional surface coatings of carboxymethyl cellulose-dopamine, provided a bioactive environment that significantly enhancedRpsattachment and biofilm formation in the presence of tomato xylem sap. Using computational approaches, we confirmed thatRpsexperienced linear increasing drag forces in xylem-mimicking channels at higher flow rates. Consistently, attachment and biofilm assays conducted in our microfluidic system revealed that both seeding time and flow rates were critical for bacterial adhesion to surface and biofilm formation inside the channels. These findings provided insights into theRpsattachment and biofilm formation processes, contributing to a better understanding of plant-pathogen interactions during wilt disease development.