The freshwater microbial community in a recreational area of Xochimilco, México was investigated and compared based on spatial (three different sites) and temporal (dry and rainy seasons) environmental variables. Many of the 16S- and 18S rRNA gene sequences recovered by DGGE fingerprinting analysis were related to phototrophic microbial phylotypes of known identity. Our genetic and morphological analysis indicated the ubiquitous presence of the microeukaryotic green algae Desmodesmus- Scenedesmus spp. and of the unicellular cyanobacteria Cyanobium spp. as the most representative populations in the samples. While 18S rRNA-DGGE fingerprinting analysis revealed a homogeneous community composition across sites and seasons, the 16S rRNA showed significant differences between localities and seasons. None of the cyanobacteria species with potential to produce toxins were identified across the investigated samples. Correlations between biotic and abiotic variables evidenced an important difference between the dry and the rainy season, with a greater consistency in data from the rainy season. According to Principal Component Analysis (PCA), a strong relation between inorganic nitrogen, species richness, and subaquatic irradiance determines environmental variability in Xochimilco. Complementary and relevant data in results obtained from microscopy, fingerprinting, and statistical analysis applied in ecology indicate that a multifaceted approach to the study of microbial communities is necessary to accomplish a comprehensive scientific framework and to generate proper management strategies.