The strain-promoted alkyne-azide cycloaddition (SPAAC) reaction can be used to modify the surface of bacteria for a variety of applications including drug delivery, biosensing, and imaging. This is usually accomplished by first installing a small azide group within the peptidoglycan and then delivering exogenous cargo (e.g., a protein or nanoparticle) modified with a cyclooctyne group, such as dibenzocyclooctyne (DBCO), for conjugation. However, DBCO is comparatively bulky and hydrophobic, increasing the propensity of some payloads to aggregate. In this study, we sought to invert this paradigm by exploring two novel strategies for incorporating DBCO into the peptidoglycan of and compared them to an established approach using DBCO-vancomycin. We demonstrate that DBCO-modified small molecules belonging to all three classes─a sortase peptide substrate (LPETG), two d-alanine derivatives, and vancomycin─can selectively label the surface to varying degrees. In contrast to DBCO-vancomycin, the DBCO-d-alanine variants do not adversely affect the growth of or lead to off-target labeling or toxicity in HEK293T or RAW 264.7 cells. Finally, we show that, unlike IgG3-Fc labeled with DBCO groups, IgG3-Fc labeled with azide groups is stable (i.e., remains water-soluble) under normal storage conditions, retains its ability to bind the immune receptor CD64, and can be successfully attached to the surface of DBCO-modified . We believe that the labeling strategies explored herein will expand the paradigm of specific, nontoxic SPAAC-mediated labeling of the surface of and other Gram-positive bacteria, opening the door for new applications using azide-modified cargo.