Production of reactive oxygen species (ROS) is a hallmark of successful recognition of infection and activation of plant defenses. ROS play multifaceted signaling functions mediating the establishment of multiple responses and can act as local toxins. Controversy surrounds the origin of these ROS. Several enzymatic mechanisms, among them a plasma membrane NADPH oxidase and cell wall peroxidases, can be responsible for the ROS detected in the apoplast. However, high levels of ROS from metabolic origins and/or from downregulation of ROS-scavenging systems can also accumulate in different compartments of the plant cell. This compartmentalization could contribute to the specific functions attributed to ROS. Additionally, ROS interact with other signals and phytohormones, which could explain the variety of different scenarios where ROS signaling plays an important part. Interestingly, pathogens have developed ways to alter ROS accumulation or signaling to modify plant defenses. Although ROS have been mainly associated with pathogen attack, ROS are also detected in other biotic interactions including beneficial symbiotic interactions with bacteria or mycorrhiza, suggesting that ROS production is a common feature of different biotic interactions. Here, we present a comprehensive review describing the newer views in ROS signaling and function during biotic stress.