Efficient and reproducible production of mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) is essential for their therapeutic application. In this study, we investigated, for the first time, the impact of long-term serum-free culture of bone marrow-derived MSCs (BMMSCs) on EV production, physicochemical properties, and biological activity. EV yield exhibited a time-dependent pattern, increasing during the early to mid-conditioning phases (96–192 h) and declining at later time points, correlating with reduced cell density. Despite these variations in quantity, EVs maintained a consistent nanoscale size distribution, stable zeta potential, and typical vesicular morphology throughout the culture period. Molecular characterisation confirmed the stable expression of classical EV markers (CD9, CD63, and CD81), indicating preserved EV identity during extended culture. Functional assays further demonstrated that EVs isolated at all time points retained their biological activity, promoting cell proliferation, migration, and immunomodulation. These findings suggest that although EV production is influenced by the metabolic state and abundance of parental cells, prolonged serum-free culture does not compromise EV structural integrity, molecular composition, or functional properties. Overall, long-term BMMSC culture primarily affects EV yield rather than quality, supporting the feasibility of extended harvesting strategies while highlighting the importance of optimising collection timing for efficient EV production.