In the food industry, synthetic preservatives are commonly used to inhibit microbial spoilage, prevent chemical and nutritional changes, and extend shelf life. However, growing consumer demand for healthier, minimally processed foods without synthetic additives has lead toto a search for natural preservation alternatives, such as antimicrobial peptides from yeasts. Pathogenic microorganisms such as Escherichia coli, Listeria monocytogenes, and Salmonella spp. represent major contamination risks to food products. To address these risks, various bioactive compounds with antimicrobial properties, including antimicrobial peptides, bacteriocins, and mycocins, have been identified as potential natural preservatives. This study aimed to assess the antimicrobial efficacy of peptide fractions from Saccharomyces cerevisiae, Wickerhamomyces anomalus, and Tetrapisispora phaffii against E. coli (ATCC 25922), L. monocytogenes (ISA 4008), and Salmonella spp. (ISA 4008). MIC tests revealed varying levels of activity, with peptides from S. cerevisiae demonstrating the strongest effect, achieving an MIC of 250 µg/mL against all three pathogens. Peptides from W. anomalus showed MICs of 2300 µg/mL for both E. coli and L. monocytogenes, and 1150 µg/mL against Salmonella spp. In contrast, peptides from T. phaffii required 1250 µg/mL to inhibit E. coli and Salmonella spp., and 2500 µg/mL to inhibit L. monocytogenes, indicating differing levels of antimicrobial efficacy. Additionally, a challenge test was conducted to determine the shelf life of the juice. The objective was to assess the antimicrobial efficacy of yeast-derived peptides for preserving watermelon juice at 25°C. The results showed that S. cerevisiae peptides achieved a >5-log CFU/mL reduction in E. coli within seven days, reducing the pathogen to undetectable levels and suggesting irreversible cellular damage. Peptides from W. anomalus also exhibited antimicrobial effects, though less pronounced, with a reduction of 1.1 log CFU/mL. These findings suggest that yeast-derived peptides, particularly from S. cerevisiae, could serve as effective natural biopreservatives to enhance the microbial safety of fruit juices.