The killer systems of S. cerevisiae are defined by the co-infection of two viral agents, an M virus and a helper virus. Each killer toxin is determined by the type of M virus (ScV-M1, ScV-M2, ScV-M28, and ScV-Mlus), which encodes a specific toxin (K1, K2, K28, and Klus). Since their discovery, interest in their potential use as antimicrobial agents has driven research into the mechanisms of action of these toxins on susceptible cells. This review provides an overview of the key aspects of killer toxins, including their origin and the evolutionary implications surrounding the viruses involved in the killer system, as well as their potential applications in the biomedical field and as a biological control strategy. Special attention is given to the mechanisms of action described to date for the various S. cerevisiae killer toxins.