Research on biologically active compounds has been increased in order to improve plant protection against various environmental stresses. Among natural sources, plants are the fundamental material for studying these bioactive compounds as their immune system consists of many peptides, proteins, and hormones. Osmotin is a multifunctional stress-responsive protein belonging to pathogenesis-related 5 (PR-5) defense-related protein family, which is involved in inducing osmo-tolerance in plants. In this scenario, the accumulation of osmotin initiates abiotic and biotic signal transductions. These proteins work as antifungal agents against a broad range of fungal species by increasing plasma membrane permeability and dissipating the membrane potential of infecting fungi. Therefore, overexpression of tobacco osmotin protein in transgenic plants protects them from different stresses by reducing reactive oxygen species (ROS) production, limiting lipid peroxidation, initiating programmed cell death (PCD), and increasing proline content and scavenging enzyme activity. Other than osmotin, its homologous proteins, osmotin-like proteins (OLPs), also have dual function in plant defense against osmotic stress and have strong antifungal activity.