ABSTRACT The antibiotic-tolerant biofilms present in tuberculous granulomas add an additional layer of complexity when treating mycobacterial infections, including tuberculosis (TB). For a more efficient treatment of TB, the biofilm forms of mycobacteria warrant specific attention. Here, we used Mycobacterium marinum (Mmr) as a biofilm-forming model to identify the abundant proteins covering the biofilm surface. We used biotinylation/streptavidin-based proteomics on the proteins exposed at the Mmr biofilm matrices in vitro to identify 448 proteins and ex vivo proteomics to detect 91 Mmr proteins from the mycobacterial granulomas isolated from adult zebrafish. In vitro and ex vivo proteomics data are available via ProteomeXchange with identifiers PXD033425 and PXD039416 , respectively. Data comparisons pinpointed the molecular chaperone GroEL2 as the most abundant Mmr protein within the in vitro and ex vivo proteomes, while its paralog, GroEL1, with a known role in biofilm formation, was detected with slightly lower intensity values. To validate the surface exposure of these targets, we created in-house synthetic nanobodies (sybodies) against the two chaperones and identified sybodies that bind the mycobacterial biofilms in vitro and those present in ex vivo granulomas. Taken together, the present study reports a proof-of-concept showing that surface proteomics in vitro and ex vivo proteomics combined is a valuable strategy to identify surface-exposed proteins on the mycobacterial biofilm. Biofilm surface–binding nanobodies could be eventually used as homing agents to deliver biofilm-targeting treatments to the sites of persistent biofilm infection. IMPORTANCE With the currently available antibiotics, the treatment of TB takes months. The slow response to treatment is caused by antibiotic tolerance, which is especially common among bacteria that form biofilms. Such biofilms are composed of bacterial cells surrounded by the extracellular matrix. Both the matrix and the dormant lifestyle of the bacterial cells are thought to hinder the efficacy of antibiotics. To be able to develop faster-acting treatments against TB, the biofilm forms of mycobacteria deserve specific attention. In this work, we characterize the protein composition of Mmr biofilms in bacterial cultures and in mycobacteria extracted from infected adult zebrafish. We identify abundant surface-exposed targets and develop the first sybodies that bind to mycobacterial biofilms. As nanobodies can be linked to other therapeutic compounds, in the future, they can provide means to target therapies to biofilms.