Biosurfactants are amphiphilic biocompounds produced by microorganisms, recognized for their surface-active properties and broad biotechnological applicability. The rising concern over antimicrobial resistance and environmental impact of synthetic chemicals has increased the demand for natural and eco-friendly alternatives. Biosurfactants, due to their unique chemical structure and multifunctional properties, have emerged as promising candidates in this regard. These compounds have gained increasing attention due to their biodegradability, low toxicity, and potential to replace synthetic surfactants in various industries. In particular, their antimicrobial properties make them promising agents for applications in food safety, pharmaceuticals, and environmental protection. Pseudomonas fluorescens, a well-known biosurfactant-producing bacterium, has been extensively studied for its capacity to produce rhamnolipids with antimicrobial activity. Therefore, this study aimed to assess the antibacterial potential of the biosurfactant synthesized by Pseudomonas fluorescens ICCF 392 against Bacillus cereus, a Gram-positive bacterium frequently associated with foodborne illnesses. The biosurfactant was obtained through submerged fermentation and partially purified. The antibacterial activity was evaluated using the agar well diffusion method, which revealed a clear zone of inhibition measuring 20 mm in diameter. These findings indicate that microbial biosurfactants can serve as effective and sustainable alternatives to conventional antimicrobial agents. Further studies will focus on detailed characterization of the biosurfactant, its spectrum of activity, and formulation potential in various delivery systems.