Extracellular vesicles (EVs), lipid bilayer nanovesicles secreted by cells, carry nucleic acids, proteins, and other bioactive molecules that influence recipient cells and modulate various biological processes. This study investigated how energy depletion and fermentation processes influence the characteristics and physiological functions of EVs secreted by <i>Saccharomyces cerevisiae</i>. Specifically, we analyzed EVs derived from 24-h cultures, representing the glucose utilization phase, and 72-h cultures, representing the starvation stage. Under energy-depleted conditions (72-h cultures), yeast secreted a higher number of EV particles, albeit with a smaller average particle size. In contrast, EVs from yeast cultured for 24 h, during the glucose utilization phase, were enriched in Pep12-rich endosome-derived vesicles and exhibited 71% higher cellular internalization efficiency. Proteomic and transcriptomic analyses revealed distinct protein and microRNA profiles between EVs from 24- and 72-h cultures, highlighting their potential roles in tissue regeneration, cell proliferation, and collagen synthesis. As a result, EVs derived from 24-h cultures exhibited a 15% greater effect in promoting collagen synthesis. The differential effects on collagen production may be attributed to the efficiency of endocytosis and the specific protein and microRNA cargo of the EVs. This study emphasizes the functional potential and unique properties of yeast-derived EVs while proposing strategies to modulate EV composition by adjusting the yeast culture duration and the energy source in the medium. Further research is needed to control yeast-produced EV components and to understand their mechanisms of action for effective therapeutic applications.