The ability to differentiate microorganisms using pyrolysision trap mass spectrometry was demonstrated for five Gram-negative disease-causing organisms: Brucella melitensis, Brucella suis, Vibrio cholera, Yersinia pestis, and Francisella tularensis. Bacterial profiles were generated for gamma-irradiated bacterial samples using pyrolytic methylation and compared for electron ionization and chemical ionization using several liquid reagents with increasing proton affinities. Electron ionization combined with pyrolysis caused extensive fragmentation, resulting in a high abundance of lower mass ions and diminishing the diagnostic value of the technique for compound identification and bacterial profiling. Chemical ionization reduced the amount of fragmentation due to ionization while enhancing the molecular ion region of the fatty acids. As the proton affinity of the reagent increased, the protonated molecular ions of the fatty acids became the predominant ions observed in the mass spectrum. As a result, chemical ionization was shown to be more effective than electron ionization in bacterial profiling. Whereas the bacteria could be distinguished at the Genera level using electron ionization, further differentiation to the subspecies level was possible using chemical ionization. The greatest separation among the five test organisms, in terms of Euclidean distances, was obtained using ethanol as the chemical ionization reagent and using pooled masses representing specific fatty acid biomarkers rather than total ion profiles.