The review summarises recent advances toward a greater comprehensive assessment of microbial diversity in aquatic environments using the fatty acid methyl esters (FAME) and phospholipid fatty acids (PLFA) approaches. These methods are commonly used in microbial ecology because they do not require the culturing of microorganisms, are quantitative and reproducible and provide valuable information regarding the structure of entire microbial communities. Because some fatty acids are associated with taxonomic and functional groups of microorganisms, they allow particular groups of microorganisms to be distinguished. The integration of fatty acid-based methods with stable isotopes, RNA and DNA analyses enhances our knowledge of the role of microorganisms in global nutrient cycles, functional activity and phylogenetic lineages within microbial communities. Additionally, the analysis of fatty acid profiles enables the shifts in the microbial diversity in pristine and contaminated environments to be monitored. The main objective of this review is to present the use of lipid-based approaches for the characterisation of microbial communities in water columns, sediments and biomats.