Microbial mats are stratified microbial communities composed by highly inter-related populations and therefore are frequently chosen as model systems to study diversity and ecophysiological strategies. The present study describes an integrated approach to analyze microbial quinones and intact polar lipids (IPLs) in microbial mats within layers as thin as 500 microm by liquid chromatography-tandem mass spectrometry. Quinone profiles revealed important depth-related differences in community composition in two mat systems. The higher abundance of ubiquinones, compared to menaquinones, reflected the clear predominance of microorganisms belonging to aerobic alpha-, beta-, and gamma-Proteobacteria in Ebro delta estuarine mats. Hypersaline photosynthetic Camargue mats (France) showed a predominance of menaquinone-9 at the top of the mat, which is consistent with an important contribution of facultative aerobic or anaerobic bacteria in its photic zone. Quinone indices also indicated a higher diversity of non-phototrophs and a more anaerobic character in the hypersaline mats. Besides, the dissimilarity index suggested that the samples were greatly influenced by a depth-related redox state gradient. In the analysis of IPLs, there was a predominance of phosphatidylglycerols and sulfoquinovosyldiacylglycerols, the latter being an abundant biomarker of Cyanobacteria. This combined approach based on quinone and IPL analysis has proven to be a useful method to establish differences in the microbial diversity and redox state of highly structure microbial mat systems at a fine-scale level.