Mycobacterium tuberculosis, the etiological agent of human tuberculosis, requires the non-mammalian disaccharide trehalose for growth and virulence. Recently, detectable trehalose analogues have gained attention as probes for studying trehalose metabolism and as potential diagnostic imaging agents for mycobacterial infections. Of particular interest are deoxy-[(18)F]fluoro-d-trehalose ((18)F-FDTre) analogues, which have been suggested as possible positron emission tomography (PET) probes for in vivo imaging of M. tuberculosis infection. Here, we report progress toward this objective, including the synthesis and conformational analysis of four non-radioactive deoxy-[(19)F]fluoro-d-trehalose ((19)F-FDTre) analogues, as well as evaluation of their uptake by M. smegmatis. The rapid synthesis and purification of several (19)F-FDTre analogues was accomplished in high yield using a one-step chemoenzymatic method. Conformational analysis of the (19)F-FDTre analogues using NMR and molecular modeling methods showed that fluorine substitution had a negligible effect on the conformation of the native disaccharide, suggesting that fluorinated analogues may be successfully recognized and processed by trehalose metabolic machinery in mycobacteria. To test this hypothesis and to evaluate a possible route for delivery of FDTre probes specifically to mycobacteria, we showed that (19)F-FDTre analogues are actively imported into M. smegmatis via the trehalose-specific transporter SugABC-LpqY. Finally, to demonstrate the applicability of these results to the efficient preparation and use of short-lived (18)F-FDTre PET radiotracers, we carried out (19)F-FDTre synthesis, purification, and administration to M. smegmatis in 1 hour.