Probiotics, known as live microorganisms, have been shown to have a beneficial effect on health at appropriate doses. Recently, a probiotic mixture containing eight live bacterial strains, VSL#3, has attracted considerable interest due to its combined effect. VSL#3 is the only probiotic considered a food for special medical purposes; it primarily participates in regulating intestinal barrier function, improving the function of tight junction proteins, balancing the composition of the gut microbiota, and regulating the expression of immune-related cytokines, among other effects. The aim of this review is to analyze the mechanism of action and treatment with VSL#3 in chronic diseases in animals and humans (including children). Numerous studies have demonstrated that VSL#3 has a therapeutic or preventive effect in various systemic diseases, such as digestive diseases (gastrointestinal and hepatic), obesity and diabetes, allergic diseases, neurological diseases, atherosclerosis, bone diseases, and diseases of the female reproductive system.