Pierce's disease (PD) of grapevines is caused by the xylem-limited bacterium Xylella fastidiosa (Xf). All Vitis vinifera-based cultivars are susceptible to Xf infection; however, many grape species from the southern United States (such as V. arizonica, V. shuttleworthii, V. simpsonii, V. smalliana, and Muscadinia rotundifolia) are resistant. In this study, the effects of xylem sap from PD-resistant and PD-susceptible grapes, as well as several free cell wall constituents, on in vitro bacterial growth, biofilm formation, and cellular aggregation were investigated. Media containing xylem sap from PD-susceptible plants provided better support for bacterial growth and biofilm formation than media supplemented with xylem sap from PD-resistant plants. Culturing Xf on media containing various purified cell wall constituents demonstrated that CM-cellulose, xylan, beta-D-glucan, k-carrageenan, cello-oligosaccharide and laminarin promoted bacterial growth whereas lichenan suppressed growth. However, only laminarin, xylan, and k-carrageenan promoted biofilm formation in vitro. Lichenan, oligosaccharide, k-carrageenan, laminarin, xylan and beta-D-glucan all significantly decreased Xf cellular aggregation in vitro. This study suggests that differences in xylem sap composition and cell wall properties among PD-resistant and PD-susceptible grapes may affect characteristics of Xf growth, biofilm formation and cellular aggregation involved in pathogenesis.