Tacrolimus, a 23-membered polyketide macrolide, is isolated as a metabolite from the whole fermentation broth of Streptomyces species. This potent immunosuppressive calcineurin inhibitor has been widely used in various fields of medicine, including transplantology, dermatology and pharmacotherapy of autoimmune diseases. The current study was focused on optimisation of tacrolimus biosynthesis by Streptomyces tsukubaensis through the use of culture media supplements. Enrichment of the fermentation medium with propionic acid, propylene glycol or propanol resulted in a 5.5-, 3.5- and 1.8-fold, respectively, increase in tacrolimus production. The optimal concentration of the precursors was 0.25% for both propanol and propionic acid and 0.75% for propylene glycol. The mode of action of each media supplement tested was unique. For instance, propionic acid acted as a tacrolimus biosynthesis precursor while propylene glycol induced mycelial growth of S. tsukubaensis. Results from the current study clearly demonstrate that a set of novel culture medium supplements considerably increased tacrolimus production. Application of such promoters of tacrolimus biosynthesis may lead to a substantial improvement in the production of tacrolimus by S. tsukubaensis in industrial fermentation processes.