Extracellular vesicles (EVs) can transfer antigens and immunomodulatory molecules, and such EVs released by antigen-presenting cells equipped with immunostimulatory functions have been utilized for vaccine formulations. A prior high-throughput screening campaign led to the identification of compound 634 (1), which enhanced EV release and increased intracellular Ca2+ influx. Here, we performed systematic structure-activity relationship (SAR) studies to investigate the scaffold for its potency as a vaccine adjuvant. Synthesized compounds were analyzed in vitro for CD63 reporter activity (a marker for EV biogenesis) in human THP-1 cells, induction of Ca2+ influx, IL-12 production, and cell viability in murine bone-marrow-derived dendritic cells. The SAR studies indicated that the ester functional group was requisite, and the sulfur atom of the benzothiadiazole ring replaced with a higher selenium atom (9f) or a bioisosteric ethenyl group (9h) retained potency. Proof-of-concept vaccination studies validated the potency of the selected compounds as novel vaccine adjuvants.