How cues that trigger the wound response result in tissue healing is a question of immense biological and medical importance. Here we uncover roles for mitochondrial reactive oxygen species (mtROS) during Drosophila dorsal closure, a model for wound healing. By using real-time visualization of ROS activity and single-cell perturbation strategies, we demonstrate that stochasticities in ROS generation in the amnioserosa are necessary and sufficient to trigger cell delamination. We identify dose-dependent effects of mtROS on actomyosin and mitochondrial architecture, dynamics, and activity that mediate both stochasticities in cell behavior and the phases of tissue dynamics accompanying dorsal closure. Our results establish that ROS levels tune cell behavior and tissue dynamics qualitatively and quantitatively. They identify a pathway triggered by ROS and mediated by the Rho effector ROCK and its substrates that influences tissue patterning and homeostasis through the coordinate regulation of both mitochondrial morphology and tissue tension.