At present, biodiesel is primarily comprised of fatty acid methyl esters (FAMEs) obtained by the transesterification of a variety of vegetable oils with methanol. However, transesterification can be carried out with a wide variety of alcohols other than methanol. This paper presents experimental studies carried out on a diesel engine supplied with a range of single-molecule fuels to investigate the effect of fuel molecular structure on combustion and emissions. Eight fatty acid esters that can be produced by transesterification of vegetable oils and animal fats using methanol, ethanol, n-propanol, n-butanol, iso-propanol, iso-butanol, and tert-butanol were investigated. The aim was to ascertain the way in which the structure of the alcohol reactant and the corresponding moiety in the ester molecule affects combustion and emissions. Previously obtained experimental results from a second set of 18 nonoxygenated acyclic hydrocarbons were used to assist the interpretation of the test results from the eight fatty acid esters. The engine tests were carried out at constant injection timing, and they were repeated at constant ignition timing and at constant ignition delay, the latter being achieved through the addition to the various fuels of small quantities of ignition improver (2-ethyl hexyl nitrate). Only small differences in ignition quality of the fatty acid esters were found with variation of the molecular structure of the alcohol moiety. Where present, differences in ignition delay were found to be the main driver of both combustion phasing and emissions production. With the effect of ignition delay isolated, physical properties of the esters, such as thermal degradation prior to vaporization and boiling point, were found to exert an influence on levels of particulate and NOx emissions.