Indigenous lactic acid bacteria-producing bacteriocins could enhance the microbiological safety of traditional cheeses. This study aimed to detect bacteriocinogenic strains among 35 Lactobacillus plantarum isolated from artisanal, raw sheep-milk cheeses. The isolates were identified by API 50 CHL and species-specific PCR. As displayed by agar well-diffusion assay, the cell-free supernatant of L. plantarum LpU4 showed the highest antimicrobial activity against Enterococcus faecalis JH2-2. The activity was not detected after treatment with proteinase K, pepsine and pronase. An active peptide band was shown by tricine–sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis and subsequent bioassay. MALDI-TOF analysis revealed a protein of 4,866.7 Da, having no homology with other known bacteriocins. The novel bacteriocin (named plantaricin LpU4) was heat stable (121 °C for 15 min), unaffected by chemicals as Tween 20, SDS, Triton ×100, EDTA, NaCl and exposure to a wide range of pH. The synthetic machinery encoding for the plantaricin was plasmid-located, as deduced by plasmids curing. Activity spectrum included several lactic acid bacteria and Staphylococcus aureus strains having antibiotic resistance phenotype. Plantaricin LpU4 showed a bacteriostatic mode of action and an enhanced activity at acidic pHs. Maximal production (3,200 AU.mL⁻¹) was reached during the late exponential phase of growth of L. plantarum LpU4. Relatively low temperature (15–25 °C) and initial pH between 5.5 and 6.2 increased the production. Since L. plantarum LpU4 produced the bacteriocin under experimental conditions mimicking the cheese environment, it could be considered a promising candidate for use as biopreservative in traditional cheese.