The present study aims to evaluate the antimicrobial and antibiofilm properties of the biosurfactants from Lactiplantibacillus pentosus MSCIN-24 (GenBank Ac. No.: MK397507) and L. pentosus No.: MK397508) against food spoilage and topical pathogens. The extracellular biosurfactants were produced in static broth culture at pH 6.8 ± 2, 37°C for 48 h and harvested by ethanol precipitation. The biosurfactants from these strains had the highest E 24 values of 100% and 93.75% in olive oil, respectively at a minimum dose of 50 mg/ml and were thermostable and enzyme stable. These glycolipid biosurfactants had broad-spectrum antimicrobial activity against the food and topical pathogens with MIC and antibiofilm values in the range of 5 to 15 mg/ml and caused cell disruption. Thus, these broadspectrum antibiofilm glycolipid biosurfactants could be used as potent antimicrobials against biofilm-forming food spoilage and topical pathogens. Practical applicationsBiofilm formation by food spoilage and topical pathogens are an important concern in the food processing industries for the health and hygiene issues of processed foods.Many commercial synthetic food preservatives show ineffective to combat these microorganisms. The biosurfactants from the food-grade Lactiplantibacillus pentosus MSCIN-24 and MSCIN-25 show promising antimicrobial and antibiofilm properties against the food spoilage and topical pathogens that could be exploited in food processing and preservation to extend their shelf life and better hygiene.