Extracellular vesicles (EVs) are produced by various cells and exist in most biological fluids. They play an important role in cell–cell signaling, immune response, and tumor metastasis, and also have theranostic potential. They deliver many functional biomolecules, including DNA, microRNAs (miRNA), messenger RNA (mRNA), long non-coding RNA (lncRNA), lipids, and proteins, thus affecting different physiological processes in target cells. Decreased immunogenicity compared to liposomes or viral vectors and the ability to cross through physiological barriers such as the blood–brain barrier make them an attractive and innovative option as diagnostic biomarkers and therapeutic carriers. Here, we highlighted two types of cells that can produce functional EVs, namely, mesenchymal stem/stromal cells (MSCs) and regulatory T cells (Tregs), discussing MSC/Treg-derived EV-based therapies for some specific diseases including acute respiratory distress syndrome (ARDS), autoimmune diseases, and cancer.