In this experimental study, spray and combustion characteristics of a single cylinder optical engine were evaluated by varying the fuel injection pressure (FIP) (40, 80, and 120 MPa). Karanja, Jatropha, and waste cooking oil (WCO) biodiesels were the test fuels and their results were compared with baseline mineral diesel. There was no significant difference observed in the spray tip penetration amongst all test fuels, however the spray cone angles of biodiesels were slightly higher than baseline mineral diesel. Mineral diesel showed relatively shorter injection delay compared to biodiesels at 40 and 80 MPa FIP. Jatropha and Karanja biodiesels showed higher flame luminosity at all FIPs, while WCO biodiesel showed lower flame luminosity, especially at higher FIPs of 80 and 120 MPa, primarily due to lower viscosity of WCO biodiesel. Flame spatial fluctuation (FSF) and flame nonhomogeneity (FNH) were also found to be higher for biodiesels at lower FIP of 40 MPa. Karanja and Jatropha biodiesels showed higher FSF and FNH at higher FIPs compared to WCO biodiesel.