This study aims to assess the microscopic characteristics of Jatropha, Karanja, and Waste cooking oil-based biodiesels vis-a-vis conventional diesel under different ambient conditions in order to understand the in-cylinder processes, while using biodiesels produced from different feedstocks in the compression ignition engines. All test-fuels were injected in ambient atmosphere using a common-rail direct injection (CRDI) fuel injection system at a fuel injection pressure (FIP) of 40 MPa. Microscopic spray characteristics were measured using phase Doppler interferometer (PDI) in the axial direction of the spray at a distance of 60–90 mm downstream of the nozzle and at 0 to 3-mm distance from the central axis in the radial direction. All biodiesels exhibited relatively larger Sauter mean diameter (SMD) of the spray droplets and higher droplet velocities compared to baseline mineral diesel, possibly due to relatively higher fuel viscosity and surface tension of biodiesels. It was also observed that SMD of the spray droplets decreased with increasing distance in the radial and axial directions and the same trend was observed for all test-fuels.