Increasing petroleum prices, increasing threat to the environment from exhaust emissions and global warming have generated intense international interest in developing renewable and alternative non-petroleum fuels for engines. Evolving technology and a recurring energy crisis necessitates a continuous investigation into the search for sustainable and clean-burning renewable fuels. In this paper, cottonseed oil methyl ester (COME) was used in a four-stroke, single-cylinder variable compression ratio diesel engine. Tests were carried out to study the effects of fuel injection timing, fuel injector opening pressure (IOP) and injector nozzle geometry on the performance and combustion of COME biodiesel fuel used in a compression ignition engine with a single fuel mode. Fuel injection timing varied from 19° to 27° before top dead centre (bTDC) in incremental steps of 4° bTDC; fuel IOP varied from 210 to 240 bar in incremental steps of 10 bar. Fuel nozzle injectors with three, four and five holes, each of 0.3 mm size, were selected for the study. The results suggested that with retarded injection timing of 19° bTDC, increased IOP of 230 bar and a four-hole nozzle injector of 0.3 mm size resulted in overall better engine performance with an increased brake thermal efficiency and reduced HC, CO and smoke emission levels.