In situ supercritical fluid derivatization and extraction was used as a sample preparation technique for the classification of bacteria using fatty acid profiling. Addition of a quaternary ammonium salt such as phenyltrimethylammonium hydroxide under static supercritical conditions directly to lyophilized, whole-cell bacteria in an extraction vessel resulted in the saponification of the bacterial lipids and derivatization of their fatty acids. The derivatized fatty acid methyl esters (FAMEs) were then extracted with supercritical CO2 and analyzed without additional treatment using GC/MS. Iso and anteiso C15:0 and C17:0 along with C18:0 were predominant in Gram-positive bacteria, while C16:1, C16:0, C18:1, and cyclopropyl cyC17:0 and cyC19:0 were significant in Gram-negative bacteria. Application of principal components analysis to the FAME GC/MS data resulted in the differentiation between Gram-positive and Gram-negative type bacteria. Differentiation between species among the same genera was also observed.