The transport cycle for RND efflux pumps, such as MtrD, remains an unresolved puzzle. This study aimed to capture the molecular details of the MtrD transport cycle, substrate binding and export mechanism of a range of antimicrobial compounds known to be transported by MtrD. As the precise mechanism of transport and the chemical basis of substrate specificity has not been fully characterized for MtrD, initial work focused on the current prophylactics’ Azithromycin, Oxacillin, TX-100, Rifampicin, Erythromycin, Novobiocin, Nonoxynol-9 and Ceftriaxone. The research also attempted to capture the role of H+-relay residues titratable residues in transport cycle. Spontaneous binding atomistic simulations were performed in triplicate over 500 ns for all the drugs, with varying charge states on the H+-relay residues (D405 & D406) of the MtrD trimer. The key results underpinned that substrates entered through the periplasmic cleft and bound to the proximal binding site of the binding monomer followed by closure of the periplasmic clefts. To study if the protonation helps in transportation of substrates, the simulations were further extended for 500ns, for substrates bound to the proximal binding site by protonating the H+-relay residues in binding and deprotonating in the other two monomers. Extended simulations supported that protonation of drug bound monomer help in transportation of drugs from proximal to distal and extrusion site.