Background: Endosomally produced by eukaryotic cells, exosomes are microvesicles involved in cell-to-cell communication. Exosomes have shown a wide range of therapeutic potential as a drug or vaccine delivery system, and they are useful as biomarkers in several disease processes. Another biological function described is pathogen dissemination through host-derived molecules released during infection, thus modulating the immune response in the host. Results: This work characterizes the exosomal fraction recovered from serum of Piscirickttesia salmonis-challenged Salmo salar specimens and from the corresponding non-challenged controls. Exosomes presented a spherical morphology and particle size distribution within 50–125 nm, showing similar parameters in both groups. The mass spectrometry analysis of exosomes isolated at 14 and 21 d post-challenge showed the presence of peptides corresponding to the three proteins of Hsp70/DnaK chaperone system (DnaK, DnaJ, and GrpE). BLAST search of these peptides showed the specificity to P. salmonis. Data are available via ProteomeXchange with identifier PXD023594. Conclusions: The chaperones were found with >95% identity in the core genome when aligned to 73 genomes of P. salmonis. The proteins also showed a high degree of similarity with other microorganisms, where this system has proven to be vital for their survival under stress conditions. The presence of these three proteins in exosomes isolated from challenged fish sera calls for further study into their potential role in bacterium pathogenicity. How to cite: Muñoz C, Carmona M, Luna O, et al. Serum-isolated exosomes from Piscirickettsia salmonis-infected Salmo salar specimens enclose bacterial DnaK, DnaJ and GrpE chaperones. Electron J Biotechnol 2022;59. https://doi.org/10.1016/j.ejbt.2022.07.003.