Background Due to the detrimental effects of chemical preservatives, there has been an increasing demand for safer, healthier and natural bio-preservatives. Bacteriocins have attracted increasing interest because of their potential as natural bio-preservatives. Results We screened a large number of Bacillus thuringiensis strains and isolated one strain (B. thuringiensis P86) with antimicrobial activity against several foodborne pathogens. Three novel leaderless bacteriocins, including thucin A1, thucin A2 and thucin A3, were purified and identified from the culture supernatant of B. thuringiensis P86, whose molecular masses were 5552.02, 5578.07 and 5609.06 Da, respectively. Thucin A1 was then selected as a representative to be tested, and it exhibited potent inhibitory activity against all tested gram-positive bacteria. More importantly, thucin A1 showed stronger antimicrobial activity than nisin A against two important foodborne pathogens Bacillus cereus and Listeria monocytogenes. In addition, thucin A1 exhibited strong acid–base adaptability (pH 2–11), high endurance to heat, good stability to trypsin and pepsin, no hemolysis activity and cytotoxicity, and could effectively inhibit or eliminate Bacillus cereus and Listeria monocytogenes in skim milk. Conclusions Our findings indicate that these novel leaderless bacteriocins are potentially promising food biopreservatives.