Bacterial cell envelope glycans are compelling antibiotic targets as they are critical for strain fitness and pathogenesis yet are virtually absent from human cells. However, systematic study and perturbation of bacterial glycans remains challenging due to their utilization of rare deoxy amino l-sugars, which impede traditional glycan analysis and are not readily available from natural sources. The development of chemical tools to study bacterial glycans is a crucial step toward understanding and altering these biomolecules. Here we report an expedient methodology to access azide-containing analogues of a variety of unusual deoxy amino l-sugars starting from readily available l-rhamnose and l-fucose. Azide-containing l-sugar analogues facilitated metabolic profiling of bacterial glycans in a range of Gram-negative bacteria and revealed differential utilization of l-sugars in symbiotic versus pathogenic bacteria. Further application of these probes will refine our knowledge of the glycan repertoire in diverse bacteria and aid in the design of novel antibiotics.