Fuel spray characteristics influence combustion, which in turn has a direct impact on engine performance and emissions. Recently, there has been an increasing interest in novel castor oil biodiesel. However, few investigations have been performed that combine both numerical and experimental biodiesel spray analyses. Hence, in this paper, we aim to explore the spray behavior of castor and jatropha biodiesel by employing numerical and experimental methods under non-evaporating, varying injection, and ambient conditions. The experimental study was carried out in a control volume vessel (CVV) at high injection and ambient pressures. The fuel atomization was modelled in ANSYS Fluent using a Lagrangian/Eulerian multiphase formulation. The results revealed that the Kelvin–Helmholtz and Rayleigh–Taylor (KHRT) model coupled with the Taylor Analogy Breakup (TAB) model provide a better estimation of the penetration length (PL) and spray cone angle (SCA) compared to the KH and TAB models. On average, Jatropha biodiesel (JB-20) and castor biodiesel (CB-20) showed a 10% to 22% longer PL, 8% to 10.6% narrower spray cone angles, and 3% to 6% less spray area, respectively, compared to diesel. The numerical predictions showed that JB-20 and CB-20 had an around 24.7–48.3% larger Sauter mean diameter (SMD) and a 38.6–73.3% average mean diameter (AMD).