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Aims and Background: Natural preservatives are a viable alternative to replace chemical preservatives that have potential toxicity and carcinogenic effects., Objective: To prove the effectiveness in increasing the microbiological stability of Minas Frescal cheese with the addition of a bacteriocin obtained from Latilactobacillus sakei as a natural preservative., Methods: A new broad-spectrum bacteriocin was evaluated for its functional activity in vitro and in situ when applied in the formulation of Minas Frescal cheese. A commercial bacteriocin was used as a positive control., Results: The inhibitory action of the bacteriocin studied was confirmed, with a reduction of 42.86% in the count of coagulase-positive Staphylococcus in relation to the negative control, at the end of the 30 days of study. For the group of thermotolerant coliforms, the bacteriocin studied showed greater efficiency than the commercial preservative. In vitro analyzes showed the inhibitory action of bacteriocin, above 87% inhibition against S. aureus, E. coli and Salmonela enteritidis, and approximately 90% against Listeria monocytogenes., Conclusion: It was concluded that the bacteriocin produced by the Latilactobacillus sakei strain has great potential for application in foods such as Minas Frescal cheese., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)