Bacterial resistance to conventional antibiotics has urged us to develop alternative strategies against bacterial pathogens. Moreover, a demand for food products containing no chemical preservatives has led us to search for new alternative technologies for food preservation. Bacteriocins – ribosomally synthesized antimicrobial peptides – have been proposed as a new alternative to conventional antibiotics or chemicals for food preservation. This study describes biosynthesis and characterization of a novel leaderless bacteriocin, geobacillin 6, which was identified in the thermophilic bacteriumParageobacillus thermoglucosidasius.Its amino acid sequence shows low similarity to other bacteriocins and it is the first leaderless-type bacteriocin identified in thermophilic bacteria. Based on structure assessment, the bacteriocin forms a multi-helix bundle. Geobacillin 6 exhibits a relatively narrow antimicrobial spectrum, it is active in the μM range and against Gram-positive bacteria, mostly thermophilic species closely related to the producer strain. Bacteriocin demonstrates stability over pH 3–11 and is highly thermostable, retaining 100% of its activity after incubation at 95°C for 6 h. Geobacillin 6 has potential in the food industry and biotechnological processes where contamination with thermophilic bacteria is undesirable.