Rainfall and associated urban runoff have been linked to an increased deterioration of environmental waters, carrying several pollutants including pathogenic microorganisms. Such happens because fecal matter is washed into storm drainage pipes that are afterward released into environmental waters. Stormwater has not been extensively characterized as it is, because most studies are performed either on drainage pipes that are often impacted by sewage leakage or directly in environmental waters following a rain event. In this study, stormwater collected directly from the streets, was monitored for the presence of fecal indicator bacteria (FIB) and three potential important sources of fecal contamination in urban environments (human, cats, and dogs) in three distinct basins in Lisbon, Portugal. Stormwater was collected in sterilized plastic boxes inserted in the storm drains, therefore collecting only runoff. High concentration of fecal contamination was detected with a high percentage of the samples displayed at least one source of contamination. A strong relationship was found between the number of detected sources and the precipitation levels. Although no statistical correlation was found between the locations and the presence of FIB or source markers, the results show a trend in geographical information on the type of urban use in each basin. To the best of our knowledge, this is the first study analyzing the runoff collected directly from the streets. This study suggests that, in urban areas, stormwater runoff is highly impacted by fecal matter, not only from domestic animals but also from human origin, before any cross-contamination in the drainage system and may, by itself, pose a high risk to human health and the environment, particularly if water reuse of this water without further disinfection treatment is the final goal.