The antibiofilm activity of a glycolipid biosurfactant isolated from the marine actinobacterium Brevibacterium casei MSA19 was evaluated against pathogenic biofilms in vitro. The isolate B. casei MSA19 was a potential biosurfactant producer among the 57 stable strains isolated from the marine sponge Dendrilla nigra. The biosurfactant production was optimized under submerged fermentation. The purified glycolipid showed a broad spectrum of antimicrobial activity. Based on the minimum inhibitory concentration/minimum bactericidal concentration ratio, the glycolipid was determined as bacteriostatic. The glycolipid biosurfactant disrupted the biofilm formation under dynamic conditions. The disruption of the biofilm by the MSA19 glycolipid was consistent against mixed pathogenic biofilm bacteria. Therefore, the glycolipid biosurfactant can be used as a lead compound for the development of novel antibiofilm agents.