Comprehensive two-dimensional gas chromatography (GC×GC) offers an interesting tool for profiling bacterial fatty acids. Flow modulated GC×GC using a commercially available system was evaluated, and different parameters such as column flows and modulation time were optimized. The method was tested on bacterial fatty acid methyl esters (BAMEs) from Stenotrophomonas maltophilia by using parallel flame ionization detector (FID)/mass spectrometry (MS). The GC×GC plots obtained for a reference sample of bacterial fatty acid esters and a bacteria sample (S. maltophilia) were very similar to those obtained by using thermal modulated GC×GC. The parallel FID/MS set-up is useful since the MS allows identification and confirmation, while the FID allows comparison of the relative fatty acid composition with existing databases. The data show that flow modulated GC×GC-FID/MS method can be applied in a routine environment and offers interesting perspectives for chemotaxonomy of bacteria.