Acoustic levitation, which allows contactless manipulation of micro‐objects with ultrasounds, is a promising technique for spheroids formation and culture. This acoustofluidic technique favors cell–cell interactions, away from the walls of the chip, which leads to the spontaneous self‐organization of cells. Using this approach, we generated spheroids of mesenchymal stromal cells, hepatic and endothelial cells, and showed that long‐term culture of cells in acoustic levitation is feasible. We also demonstrated that this self‐organization and its dynamics depended weakly on the acoustic parameters but were strongly dependent on the levitated cell type. Moreover, spheroid organization was modified by actin cytoskeleton inhibitors or calcium‐mediated interaction inhibitors. Our results confirmed that acoustic levitation is a rising technique for fundamental research and biotechnological industrial application in the rapidly growing field of microphysiological systems. It allowed easily obtaining spheroids of specific and predictable shape and size, which could be cultivated over several days, without requiring hydrogels or extracellular matrix. Formation of spheroids in acoustic levitation depends weakly on the acoustic and strongly on cell–cell interactions. The dynamic of self‐organization is cell‐type dependent. Spheroids formed and cultivated in this manner for several days are viable. Cytoskeletal and cell–cell adhesion inhibitors can disrupt self‐organization. Created with Biorender.