Thiostrepton is primarily used as an antibiotic in veterinary medicine, especially in the treatment of bacterial infections in animals, such as mastitis in cattle caused by susceptible strains of Staphylococcus and Streptococcus species. It is often formulated in combination with other antimicrobials or as an intramammary infusion for localized treatment.

In research, thiostrepton has gained attention for its ability to inhibit protein synthesis in bacteria and certain eukaryotic cells by targeting the 50S ribosomal subunit. It is used as a selective agent in molecular biology, particularly in cell culture systems that employ antibiotic resistance markers.

Additionally, studies have explored thiostrepton’s potential anti-cancer properties due to its ability to suppress the activity of the FOXM1 transcription factor, which is overexpressed in many human tumors. It has also shown activity against certain protozoan parasites, including Plasmodium falciparum, the causative agent of malaria, making it a subject of interest in antiparasitic drug development.