The cell membrane or biofilm serve as permeable barrier for xenobiotics to maintain the homeostasis of cells or bacterial community. Transport systems are essential for the uptake of nutrients and substances necessary for biofilm formation, efflux of deleterious compounds, as well as communication between cells and environment. Major facilitator superfamily (MFS) represents the largest secondary transporter family and is responsible for the transport of a broad spectrum of substrates with diverse physiochemical properties by utilizing the energy stored in electrochemical gradient across the membrane. Importantly, multidrug efflux pumps belonging to the major facilitator superfamily are important contributing factors to drug resistance and biofilm formation in many clinical strains like Mycobacterium tuberculosis. This review summarized the structural properties and functions of M. tuberculosis MFS transporters, molecular mechanisms of substrates transfer, and efflux pump inhibitors for better control of biofilm-associated infections.