The yeasts constitute a large group of microorganisms characterized by the ability to grow and survive in different and stressful conditions and then to colonize a wide range of environmental and human ecosystems. The competitive traits against other microorganisms have attracted increasing attention from scientists, who proposed their successful application as bioprotective agents in the agricultural, food and medical sectors. These antagonistic activities rely on the competition for nutrients, production and tolerance of high concentrations of ethanol, as well as the synthesis of a large class of antimicrobial compounds, known as killer toxins, which showed clearly a large spectrum of activity against food spoilage microorganisms, but also against plant, animal and human pathogens. This review describes the antimicrobial mechanisms involved in the antagonistic activity, their applications in the processed and unprocessed food sectors, as well as the future perspectives in the development of new bio-drugs, which may overcome the limitations connected to conventional antimicrobial and drug resistance.