Direct infusion electrospray ionization mass spectrometry (ESI-MS) of aqueous/methanolic extracts of biodiesel samples is shown to permit simple and fast fingerprinting typification, identification of the alcohol used in the trans-esterification process, monitoring of degradation and adulteration, and screening of residual glycerin and mono-, di-, and tri-glycerides. As proof-of-principle cases, ESI-MS fingerprints in both the positive and the negative ion modes were acquired for biodiesel samples derived from soybean oil, sunflower oil, canola oil, olive oil, castor oil, jatropha curcas oil, palm oil, lard, and tallow. In the negative ion mode, characteristic sets of ions corresponding to de-protonated fatty acids were detected for each type of biodiesel, thus allowing their reliable typification via these taxonomic markers. Biodiesel degradation was also monitored by EST(-)-MS with the detection of degradation markers, mainly oxidized fatty acids. When using ESI(+)-MS, the main diagnostic ions detected were the protonated fatty esters, which reveal therefore both the oil source and the alcohol (methanol or ethanol, or other heavier alcohols) used for trans-esterification. Residual glycerin and mono-, di-, and tri-glycerides are also detected, which makes ESI-MS also applicable for laboratory and on-site screening of biodiesel quality.