Significance Remodeling of actin cytoskeleton is thought to contribute to the establishment of effective barriers at the periphery of plant cells against fungal ingress. However, there is little information on the molecular mechanisms that regulate actin remodeling during interactions with invading pathogens. Here we show that disruption of myosin motors in Arabidopsis prevents dynamic reorganizations of actin filaments, focal organelle accumulation, and delivery of cell wall defense compounds toward the pathogen penetration site. Furthermore, the quadruple knockout mutant of class XI myosins exhibits impaired penetration resistance at the cell wall and enhanced overall disease susceptibility to both adapted and nonadapted fungal pathogens. This study provides insights into how myosins regulate cellular responses that contribute to plant immunity.