The determination of fatty acids (FA) has been extensively used as a sensitive and reproducible parameter for characterizing the soil microorganism communities and to detect various environmental stresses. The aim of this study was to assess the variability of FA in urban soils, in an attempt to use it as a differentiator of urban soil usage. FA were extracted from soils of five different usages (ornamental gardens, ornamental gardens/roadsides, roadsides, parks and urban agricultural areas) in three Portuguese cities and FA concentration was determined by gas chromatography coupled to mass spectrometry (GC-MS). A total of fifteen FA have been detected and the concentration of each FA, in general, decreased in the following order: Lisbon>Estarreja>Viseu, for all the usages of urban soil. According to soil microbial indicators, the concentration of Gram-negative bacteria was lower than the concentration of Gram-positive bacteria, which together were, in turn, higher than the concentration of fungi for all the usages of urban soil and city. This study assessed the FA profiles of urban soils, which differ as a function of soil usage. The FA profile also is at the source of the inference that stress in soil microorganism communities results from the different urban environment in each city.