Mycoplasma bovis (M. bovis) manifests as diverse clinical pathologies in cattle that significantly impacts cow health and productivity worldwide. This study investigated small extracellular vesicles (sEV) from M. bovis infected dairy cows to identify protein cargo that may be used as potential diagnostic markers during bacterial latency. Small EV were isolated from serum of dairy cows positive for M. bovis infection (n = 45) and their protein cargo compared with those from serum sEV from non-infected dairy cows (n = 49).