Natural agents from microorganisms have emerged as suitable options to replace chemical preservatives in foods. In this study, the antibacterial activity of cell-free supernatant (CFS) from five native yeasts (Saccharomyces cerevisiaeLev6 and Lev30,C. pseudointermediaLev8,Candida intermediaLev9,C. parapsilosisLev15) and the referenceS. boulardiSSB, was evaluated against some indicator food pathogens. The generation of antimicrobials was reliant on strain-, and sugar-supplemented media, which supported yeast growth established at 30 °C and 200 rpm for 48 h. Treatment with proteinase K and catalase was unable to completely abolish the inhibitory effect, indicating that the active components are likely complex combinations of acids, proteins, hydrogen peroxide, and other metabolites. Although there was no impact onListeria monocytogenes, exposure to CFS and extracellular fractions obtained through precipitation with methanol (PPm) at 120 °C for 60 min significantly (p< 0.05) increased the inhibitory activity againstEscherichia coli,Salmonella enterica,Kosakonia cowanii, andStaphylococcus aureus, indicating that the inhibitory activity was stimulated by heat. Likewise, a synergistic inhibitory action againstListeriawas obtained following the pretreatment of PPm with EDTA (ethylenediaminetetraacetic acid). These activities were yeast strain-dependent, with Lev6, Lev8, and Lev30 showing the highest activity. In addition, a heat-stable low-molecular-mass molecule under 5 kDa was detected in Lev30. Further research is required to evaluate the mode of action and characterize the composition of the released molecules in the CFS in order to develop a novel biocontrol agent based on yeasts.