Tylosin phosphate is widely used in veterinary medicine as an antibiotic, particularly in livestock and companion animals. It is effective against a range of bacterial infections, especially those caused by Gram-positive bacteria and some Gram-negative organisms. Commonly, it is used to treat respiratory infections in cattle, swine, and poultry. It also plays a role in controlling chronic respiratory disease in chickens and mycoplasma infections in various animals.

In addition to therapeutic use, tylosin phosphate has been used as a growth promoter in animal feed, enhancing feed efficiency and weight gain in livestock. It is often administered through water or feed, making it practical for large-scale farming operations.

Another important application is in the treatment of diarrhea and intestinal infections in dogs and cats, particularly those caused by Campylobacter or other susceptible pathogens. Due to its anti-inflammatory and immunomodulatory properties at low doses, it is sometimes used off-label for managing certain gastrointestinal conditions such as inflammatory bowel disease in small animals.

Its use is regulated in many countries to prevent antibiotic resistance and to ensure safe withdrawal periods before animal products enter the food chain.