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The selection of an optimal antifoam is critical for efficient fermentation, as industrial agents often have detrimental side effects like growth inhibition, while some can enhance productivity. This study presents a rational approach to developing and screening novel silicone-polyol antifoam emulsions. A key finding was the discovery of selective antibacterial activity in agent 3L10, which strongly inhibited Gram-positive bacteria (especially Corynebacterium glutamicum) but not Gram-negative strains. This specificity, likely mediated by interaction with the mycolic acid layer of C. glutamicum, highlights the necessity for strain-specific antifoam testing. A comprehensive evaluation protocol—combining chemical design, cytotoxicity screening across diverse microorganisms, determination of minimum effective concentrations (MEC), and validation in model bioreactor fermentations—was established. Through this process, agent 6T80 was identified as a promising candidate. It exhibited low MEC, high emulsion stability, no cytotoxicity, and did not impair growth or recombinant protein production in B. subtilis or P. putida fermentations. The study concludes that agent 6T80 is suitable for further application in processes involving Gram-negative and certain Gram-positive hosts, whereas agent 3L10 serves as a valuable tool for studying surfactant-membrane interactions. The developed methodology enables the targeted selection of highly efficient and biocompatible antifoams for specific biotechnological processes.