Antibiotic resistant bacteria are a threat to human life. Recently, sewers have been identified as potential reservoirs. The intermittent injection of sewage into adjacent surface waters is inevitable, due to capacity limitations of the urban drainage system. Information regarding the effect to natural freshwater biofilms (NFB) due to the intermittent contaminations are scarce. Therefore, a fundamental screening is necessary. In April, we placed NFB-attachment constructions in a brook upstream and downstream from urban drainage overflow constructions. In meanwhile two sampling campaigns were conducted. The sewage and the brook water were collected to gather information about antibiotic background exposure of ciprofloxacin (CIP), clarithromycin (CLA) and doxycycline (DOX). Six months later we experimentally determined the oxygen uptake rate (OUR) of the NFB-communities after a 24 h lasting exposure with additionally dosed antibiotics. Concentrations of 0.1, 1.0 and 10.0 mg L⁻¹ were selected. CIP, CLA and DOX were individually dosed, and also in mixtures. The mean antibiotic background concentration in sewage was in a range of 575.5–1289.1 ng L⁻¹, which mainly exceeded the concentrations published in literature. The determined mean concentration in the brook was in a range of 4.6–539.0 ng L⁻¹. The first significant inhibition of the OUR with individually dosed antibiotics started mainly at a concentration of 1.0 mg L⁻¹. Antibiotics in a mixture with concentrations of 0.1 and 1.0 mg L⁻¹ were as effective as single dosed antibiotics with a concentration of 10.0 mg L⁻¹. The increased antibiotic tolerance and resistance of NFB-communities downstream of the combined sewer overflow (CSO) structure was a consequence of a severe impact due to urban drainage overflows. Hence, NFB-communities downstream of CSO-constructions are hot spots of antibiotic tolerant and resistant subpopulations and access restrictions should be announced, if an infection risk is present. The increased antibiotic tolerance and resistance of natural freshwater biofilm communities downstream of combined sewer overflow structures is a consequence of a severe impact due to urban drainage overflows.