Efflux pump-mediated drug resistance in bacteria is a common occurrence effective for the general survival of the organism. The Mycobacterium tuberculosis genome has an abundance of adenosine triphosphate (ATP) dependent cassette transporter genes but only a handful of them are documented for their contribution to drug resistance. In this study, we inspected the potential of an ABC transporter Rv1273c from M. tuberculosis as a multidrug efflux pump and a contributor to intrinsic drug resistance. Expression of Rv1273c in Escherichia coli and M. smegmatis conferred tolerance to various structurally unrelated antibiotics. Lower accumulation of fluoroquinolones in intact E. coli and M. smegmatis cells expressing the transporter implied its active efflux activity. Energy-dependent efflux by Rv1273c was observed in real time using the lipophilic dye Nile Red. Expression of Rv1273c also resulted in an increase in biofilm formation by E. coli and M. smegmatis cells. Overall, the results indicate the possibility that Rv1273c might be a multidrug transporter with a wide substrate range and a probable contributor to biofilm formation.