Microbial contamination remains a major challenge for the wine industry, particularly due to Brettanomyces/Dekkerayeasts, which produce volatile phenols that negatively affect the flavor and aroma of wine. Traditionally, sulphur dioxide (SO₂) is used to control these microorganisms; however, growing health concerns have led to increased demand for low-sulphite or “clean label” wines. This study aimed to explore natural biocontrol strategies to reduce or eliminate the use of SO₂. A total of 87 microbial isolates—58 bacteria and 29 yeasts—were obtained from spoiled wine samples and other food sources. Phenotypic characterization and partial rDNA sequencing identified eight lactic acid bacteria belonging to Pediococcus parvulus, Lentilactobacillus hilgardii, and Sporolactobacillus inulinus species and 50 strains of Bacillus spp. Among the yeasts, 20 were identified as Brettanomyces bruxellensis and 9 as Saccharomyces cerevisiae. Antimicrobial assays revealed that Bacillus subtilis, Bacillus velezensis, Bacillus sp., and Lysinibacillus sphaericus exhibited promising inhibitory activity. Although the extraction of inhibitory compounds and potential synergistic interactions among strains remain to be explored, the current results highlight the potential of natural biocontrol agents as a sustainable alternative to SO₂. Such strategies offer the dual benefit of preserving wine quality while meeting consumer demand for safer and more natural products.