Tigecycline (TGC) is a new intravenous antibiotic with broad-spectrum effect against many drug-resistant organisms. Further, TGC is new class representer of antibacterial agents, named glycylcyclines. This group has been specifically developed to overcome the two major mechanisms of tetracycline resistance: ribosomal protection and efflux. Flavonoids are a wide class of natural compounds belonging to secondary metabolites. Diosmin (main flavonoid in citrus juices) is one of the most utilized flavonoid being the active principle of many drugs especially for the treatment of various blood vessels disorders, cancer, diabetes, premenstrual syndrome and colitis. It was isolated for the first time from Scrophularia nodosa. Human serum albumin (HSA) is a well studied protein with known primary structure, and tertiary structure which is determined by X-ray crystallography. The HSA has also multiple ligand-binding sites localized in hydrophobic cavities in subdomains IIA and IIIA, called site I and site II, respectively. In this work, we examined the effect of diosmin on the TGC-HSA by fluorescence spectroscopy, synchronous spectroscopy and molecular docking simulations. The aim is to explore the ability of diosmin to bind competitively to HSA with TGC. Experimental and theoretical results showed that diosmin increased the binding affinity of TGC to HSA.