Singlet Oxygen (SO) is among the most potent reactive oxygen species, and readily oxidizes proteins, lipids, and DNA. It can be generated at the plant surface by phototoxins in the epidermis, acting as a direct defense against pathogens and herbivores (including humans). SO can also accumulate within mitochondria, peroxisomes, cytosol, and the nucleus through multiple enzymatic and non-enzymatic processes. However, the primary location of SO in plants is in the chloroplast, where it results from transfer of light energy from PhotosystemII to triplet oxygen. SO accumulates in response to diverse stresses that perturb chloroplast metabolism, and while its short half-life precludes exiting the chloroplast, it participates in retrograde signaling through the EXECUTER1 sensor, generation of carotenoid metabolites, and possibly other unknown pathways. SO thereby reprograms nuclear gene expression and modulates hormone signaling and programmed cell death. While SO signaling has long been known to regulate plant responses to high-light stress, recent literature also suggests a role in plant interactions with insects, bacteria, and fungi. The goals of this review are to provide a brief overview of SO, summarize evidence for its involvement in biotic stress responses, and discuss future directions for the study of SO in signaling and defense.