Nowadays, consumers are aware of the health concerns regarding food additives; the health benefits of “natural” and “traditional” foods, processed without any addition of chemical preservatives, are becoming more attractive. One of the alternatives to satisfy this request are bacteriocins, which are antimicrobial peptides produced by a large number of bacteria, including lactic acid bacteria, normally acting against closely related and some spoilage and disease-causing Gram-positive pathogens. For this reason they are used in several applications, among which are biopreservation, shelf-life extension, clinical antimicrobial action and control of fermentation microflora. Toxicological studies showed that nisin intake does not cause any toxic effect to humans having an estimated lethal dose of 6950 mg/kg; thus, it is one of the bacteriocins mostly applied in the food industry as antibotulinic agent in cheese and liquid eggs, sauces and canned foods. It exhibits a wide-spectrum antimicrobial action against Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus and other pathogens. Food-grade substrates such as milk or whey can be supplemented with ex situ produced bacteriocin preparations obtained by fermentation. Preparations can be added as partially purified or purified concentrates requiring specific approval as preservatives from the legislative viewpoint. Demand for new antibacterial compounds has brought great interest for new technologies able to enhance food microbiological safety. Also the dramatic rise in antibiotic-resistant pathogens has stimulated renewed efforts to identify, develop or redesign antibiotics active against multi-resistant bacteria. Numerous antibacterial agents are now being re-considered for application, among others are bacteriophages, probiotics, antimicrobial peptides and bacteriocins. To optimally exploit their desired activities, chemical or genetic engineering methods are often employed. In this review we focus on recent classification of bacteriocins, their mode of action, biotechnological applications in food and pharmaceutical industries, purification techniques and biosafety, as well as recent attempts to generate custom-designed bacteriocins using genetic engineering techniques.