Bacterial infections are major constraints in aquaculture farming. These pathogens often adapt to the biofilm mode of growth and resist antibiotic treatments. We have used a non-toxic glycolipid biosurfactant (BS-SLSZ2) derived from a marine epizootic bacterium Staphylococcus lentus to treat aquaculture associated infections in an eco-friendly manner. We found that BS-SLSZ2 contained threose, a four-carbon sugar as the glycone component, and hexadecanoic and octadecanoic acids as the aglycone components. The critical micelle concentration of the purified glycolipid was 18 mg mL-1. This biosurfactant displayed anti-adhesive activity and inhibited biofilm formation by preventing initial attachment of cells onto surfaces. The biosurfactant (at a concentration of 20 μg) was able to inhibit Vibrio harveyi and Pseudomonas aeruginosa biofilms by 80.33 ± 2.16 and 82 ± 2.03%, respectively. At this concentration, it was also able to disrupt mature biofilms of V. harveyi (78.7 ± 1.93%) and P. aeruginosa (81.7 ± 0.59%). The biosurfactant was non-toxic towards Artemia salina. In vivo challenge experiments showed that the glycolipid was effective in protecting A. salina nauplii against V. harveyi and P. aeruginosa infections. This study highlights the significance of marine natural products in providing alternative biofilm controlling agents and decreasing the usage of antibiotics in aquaculture settings.