This paper experimentally studies and numerically analyzes the spray and atomization characteristics of biodiesel and dimethyl ether (DME) fuels as alternatives to diesel fuel. To analyze the macroscopic characteristics (such as axial/radial distance and spray cone angle and area), the macroscopic spray behaviors of three fuels (diesel, biodiesel, and DME) were measured using frozen images obtained from the visualization system. For the numerical analysis, the overall Sauter mean diameter (SMD) and the SMD contour plot of three fuel sprays were calculated using the KIVA-3V code with the addition of the fuel properties in the fuel library. The macroscopic characteristics of biodiesel spray, as the axial/radial distance, spray cone angle, and spray area, show a similar trend to that of diesel spray, and DME fuel has smaller axial and radial distances than the biodiesel and diesel fuels. The biodiesel and diesel fuels were influenced by the Weber number of droplets, but the DME fuel is influenced by the Reynolds number of droplets. It was also determined that the DME spray has superior breakup performance to that of the diesel and biodiesel sprays under similar injection conditions because DME fuel has a much lower viscosity and lower surface tension and is more volatile than the other two fuels. The accumulated vapor masses of the biodiesel and diesel sprays were extremely small, but that of the DME fuel spray continually increased after the start of the injection.