In this study, a fungal species was isolated from rhizospheric soil and identified as Penicillium sp. by ITS sequencing. The Penicillium sp. has been screened for the biosurfactant production, viz., haemolytic activity, oil spreading assay and emulsification index. The biosurfactant from cell-free supernatant was extracted using acid precipitation followed by solvent-solvent extraction. The physiochemical properties of the extracted biosurfactant were analysed using FTIR; the major peaks that show at 1720 cm, 1531 cm, 1419 cm, 1251 cm and 1010 cm correspond to aliphatic chains, sugars and ester carbonyl groups. The fatty acids present in the extracted biosurfactant were analysed using GCMS, in which a molecular mass of 256 and 284 m/z showed the presence of n-hexadecenoic acid and octadecanoic acid respectively which indicate the presence of rhamnolipid, which is a major biosurfactant. The biosurfactant extracted from Penicllium sp. demonstrated antibacterial activity against Escherichia coli and Staphylococcus aureus. In future perspectives, the biosurfactant extracted from the isolated species holds great potential as a broad-spectrum antibacterial agent and could be utilized in various healthcare applications.