Mycoplasma bovis (M. bovis) is a pathogenic bacterium that causes significant production and welfare issues in cattle. The dairy industry is essential to New Zealand, contributing over a quarter of the country’s total exports. Each year, ~4.7 million cows produce ~20.5 billion litres of milk, which is worth ~$23 billion NZD to the New Zealand economy. An unregulated M. bovis outbreak would be catastrophic for the country. Eradication is challenging as M. bovis can infect host cells intracellularly, reducing detection from immunomodulatory cells. With an aim to improve diagnostics for post-eradication surveillance, small extracellular vesicles (sEVs) were used to identify novel biomarkers of M. bovis infection. Readily accessible in body fluids, these nanoparticles encapsulate protein cargo indicative of the metabolic state of their tissue of origin. Here, we developed a three-dimensional in vitro cell culture model using static bioreactors to investigate changes in host cell sEV protein cargo during M. bovis infection. Size exclusion chromatography columns were used to isolate sEVs from control and co-culture bioreactor flasks. Liquid chromatography-trapped ion mobility spectrometry-tandem mass spectrometry (LC–TIMS-MS/MS) was used to investigate the proteome of sEVs.