Effect of the presence of a scaffold mimicking in vivo situation on bubble dynamics was investigated to study the mechanisms of in vivo sonoporation. A high-speed observation system was improved to observe bubble and cell dynamics under exposure to 3-cycle pulsed ultrasound of 1 MHz in center frequency and 1.1 MPa in peak negative pressure. Observation results showed that the stiffness of the scaffold has significant effect on the bubble dynamics: a microbubble on a hard scaffold remains on its hard surface but a bubble on a soft scaffold make a translational movement in the direction separating from the soft surface. The same movement was confirmed about a bubble adhering to cell membrane surface, and bubble-cell interaction was classified into four phenomena. Higher frequencies of cell membrane damage were found in the two phenomena: a phenomenon that a bubble moving in the separating direction drags a point of the cell membrane that the bubble is adhering, and a phenomenon that a jetting flow generated by bubble-bubble interaction causes local deformation of a cell. The results suggest that efficiency of in vivo sonoporation can be improved by promoting the mechanisms observed in the two phenomena.