Abstract Sophorolipids are glycolipids that have natural antimicrobial properties and present great potential in the pharmaceutical field. The present study aimed to produce sophorolipids from Candida bombicola using a chicken fat-based medium and evaluate the antimicrobial activity against Gram-negative (Proteus mirabilis, Escherichia coli, Salmonella enterica subsp. enterica) and Gram-positive bacteria (Enterococcus faecium, Staphylococcus aureus and Streptococcus mutans). The production of sophorolipids reached 27.86 g L-1. Based on the structural characterization, 73.55% of the sophorolipids present a mixture of acidic monoacetylated C18:2 and lactonic diacetylated C16:0, and 26.45% were present in the diacetylated C18:1 lactonic form. Bacteria submitted to sophorolipid exposure showed a reduction in viability at doses of 500 μg mL-1 and 2,000 μg mL-1 against Gram-positive and Gram-negative bacteria, respectively. These results suggest that sophorolipids produced in chicken fat medium may be used as antimicrobial agents to prevent or eliminate contamination by different pathogens.