In most cities, streets are designed for collecting and transporting dirt, litter, debris, storm water and other wastes as a municipal sanitation system. Microbial mats can develop on street surfaces and form microbial communities that have never been described. Here, we performed the first molecular inventory of the street gutter-associated eukaryotes across the entire French capital of Paris and the non-potable waters sources. We found that the 5782 OTUs (operational taxonomic units) present in the street gutters which are dominated by diatoms (photoautotrophs), fungi (heterotrophs), Alveolata and Rhizaria, includes parasites, consumers of phototrophs and epibionts that may regulate the dynamics of gutter mat microbial communities. Network analyses demonstrated that street microbiome present many species restricted to gutters, and an overlapping composition between the water sources used for street cleaning (for example, intra-urban aquatic networks and the associated rivers) and the gutters. We propose that street gutters, which can cover a significant surface area of cities worldwide, potentially have important ecological roles in the remediation of pollutants or downstream wastewater treatments, might also be a niche for growth and dissemination of putative parasite and pathogens.