Fuel atomization and air-fuel mixing processes play a dominant role on engine performance and emission characteristics in a direct injection compression ignition engine. Understanding of microscopic spray characteristics is essential to predict combustion phenomena. The present work investigates the atomization and mixing characteristics of biodiesel fuels in a constant volume combustion chamber. Waste cooking oil, Jatropha, and Karanja biodiesels were applied and the results were compared with those of diesel fuel. The tested fuels were injected by a common-rail injection system with injection pressures of 40, 80, and 120 MPa. A high-speed camera with a long distance microscopic lens was utilized to capture the near nozzle flow characteristics. Sauter mean diameter (SMD) was measured by a phase doppler particle analyzer. The experimental results showed that the biodiesels had poor fuel atomization characteristics showing longer liquid tip penetration length and narrower spray angle than those of diesel. Based on microscopic imaging, biodiesels exhibited longer injection delay and resisted formation of ligaments compared to baseline diesel due to higher viscosities and surface tension. In addition, the estimation of SMD size revealed that biodiesels had larger SMD than that of baseline diesel.