The emergence of antimicrobial resistances causes serious public health concerns worldwide. In recent years, the aquatic ecosystem has been recognized as a reservoir for antibiotic-resistant bacteria and antibiotic resistance genes (ARGs). The prevalence of 11 ARGs, active against six antibiotic classes (beta-lactams, aminoglycosides, tetracycline, macrolides, trimethoprim, and sulfonamides), was evaluated at a karst spring (Gallusquelle) in Germany, using molecular biological methods. In addition, fecal indicator bacteria (FIB), turbidity, electrical conductivity, spring discharge, and microbial source tracking markers specific for human, horse, chicken, and cow were determined. The ARGs most frequently detected were ermB (42.1%), tet(C) (40.8%). sul2 (39.5%), and sul1 (36.8%), which code for resistance to macrolides, tetracycline and sulfonamides, respectively. After a heavy rain event, the increase in FIB in the spring water was associated with the increase in ARGs and human-specific microbial source tracking (MST) markers. The determined correlations of the microbiological parameters, the observed overflow of a combined sewer overflow basin a few days before the increase of these parameters, and the findings of previous studies indicate that the overflow of this undersized basin located 9 km away from the spring could be a factor affecting the water quality of the karst spring. Our results provide a scientific basis for minimization of the input of fecal pollution and thus ARGs into the karst spring. (C) 2020 Published by Elsevier B.V.