The river Dambovita, which crosses Bucharest city, is characterized by two superposed artificial riverbeds: the lower part, under Dambovita river floor, which is a channel containing sewage from the city and the upper part, which is cleaner and combines with the lower part when exiting Bucharest. This study aims to evaluate the anthropogenic impact on the water quality of the upper canal. Samples were collected from 10 points along the sector, during two sampling campaigns, between January and February 2015. These points were chosen based on the ease of access, the environmental characteristics and any possible human influence. Physico-chemical parameters were measured: the concentration and saturation of dissolved oxygen, temperature, pressure, pH, turbidity, conductivity, amount of ammonium, phosphorus and nitrates. In addition, fluorescence spectroscopy method was used to determine the presence and types of organic substances in water samples. The results showed significant differences between the urban sector of Dambovita and the final sampling point, situated downstream of the Glina wastewater treatment plant. Fluorescence measurements showed that the quantity of humic substances had a continuous increase along the sector. Regarding the microbial fraction, fluorescence spectroscopy revealed a sudden increase at the sample collected from the entrance of the river in Bucharest and at Glina sewage effluent discharge point. Fluorescence results evidenced the anthropogenic impact on the water quality of Dambovita River. In conclusion, the quality of Dambovita waters varies across space and time, depending on human influence affecting the areas from where samples were taken and also reflecting a temporal variation, with a drop in quality during January, caused by weather conditions that lead to the concentration and stagnation of pollutants.