Bioprinting could spatially align various cells in high accuracy to simulate complex and highly organized native tissues. However, the uniform suspension and low concentration of cells in the bioink and subsequently printed construct usually results in weak cell-cell interaction and slow proliferation. Acoustic manipulation of biological cells during the extrusion-based bioprinting by a specific structural vibration mode was proposed and evaluated. Both C2C12 cells and human umbilical vein endothelial cells (HUVECs) could be effectively and quickly accumulated at the center of the cylindrical tube and consequently the middle of the printed construct with acoustic excitation at the driving frequency of 871 kHz. The full width at half maximum (FWHM) of cell distributions fitted with a Gaussian curve showed a significant reduction by about 2.2 fold in the printed construct. The viability, morphology, and differentiation of these cells were monitored and compared. C2C12 cells that were undergone the acoustic excitation had nuclei oriented densely within ±30° and decreased circularity index by 1.91 fold or significant cell elongation in the printing direction. In addition, the formation of the capillary-like structure in the HUVECs construct was found. The number of nodes, junctions, meshes, and branches of HUVECs on day 14 was significantly greater with acoustic excitation for the enhanced neovascularization. Altogether, the proposed acoustic technology can satisfactorily accumulate/pattern biological cells in the printed construct at high biocompatibility. The enhanced cell interaction and differentiation could subsequently improve the performance and functionalities of the engineered tissue samples.