Bicyclomycin benzoate is primarily used as an antibiotic, particularly effective against Gram-negative bacteria. Its unique mechanism of action involves inhibiting the transcription termination factor Rho, which is crucial for bacterial RNA synthesis. This makes it a valuable tool in research for studying bacterial transcription processes.

In clinical settings, it has been explored for treating infections caused by multidrug-resistant Gram-negative pathogens, especially in cases where other antibiotics have failed. Its application extends to veterinary medicine, where it is used to combat bacterial infections in animals, ensuring health and productivity in livestock.

Additionally, bicyclomycin benzoate is utilized in microbiological studies to understand bacterial resistance mechanisms and to develop novel therapeutic strategies. Its role in inhibiting Rho factor also aids in exploring genetic regulation and expression in bacteria, contributing to advancements in molecular biology and genetics research.