The growth of undesirable microorganisms naturally present on grapes, musts, and wines is a major concern in the wine industry, often leading to considerable financial losses. Spoilage yeasts, particularly those producing volatile phenolic compounds, pose a serious threat to wine quality by altering its taste, aroma, color, and astringency. Sulfur dioxide (SO₂) is commonly used to prevent microbial contamination due to its antimicrobial and antioxidant properties. Nevertheless, its negative impact on wine sensory characteristics and potential health risks (being considered allergenic) have led to increasing regulatory restrictions. This study aimed to explore biocontrol strategies against spoilage microorganisms in wine. Eight strains of lactic acid bacteria (LAB) and 20 Brettanomyces/Dekkera yeasts were isolated from spoiled wine samples and screened for genes encoding enzymes involved in volatile phenol production. Additionally, the antimicrobial activity of microorganisms from various food matrices such as wine and elderberry was evaluated. Several microbial species demonstrated promising antagonistic activity against Brettanomyces yeasts, suggesting potential for biocontrol applications. While inhibitory substance production was evident, further concentration and purification steps are necessary. Furthermore, the strain-specific sensitivity of Brettanomyces bruxellensis must be considered in future applications. Since the sensitivity of Brettanomyces bruxellensis strains is variable, it is essential to investigate a wide range of inhibitory strains and assess their combined application to enhance the effectiveness of biocontrol strategies against this spoilage yeast.