Antibiotics are widely used in both human and veterinary medicine and antibiotic-resistant bacteria cause problems in antibiotic therapy. The current study was conducted in the catchment area of the river Swist (Germany) and focuses on the resistance of environmental Rhodospirillaceae to antibiotics used in human medicine. The samples collected reflect different levels of human impact on the environment. In total, 614 isolates were tested for antibiotic susceptibility. About half of these isolates were susceptible to all substances tested. Oxacillin resistance was observed most frequently (41%). Resistant Rhodospirillaceae were detected in wastewater effluent from a municipal sewage treatment plant, as well as in non-polluted upper reaches. The highest multi-resistance level was detected in small tributaries and it surprisingly decreased with an increasing influence of municipal wastewater. It could be shown that the detected resistances were acquired rather than intrinsic. Besides natural occurrence of multi-resistance among non-sulphur purple bacteria, horizontal gene transfer and acquired cross-resistance against veterinary antibiotics are assumed to be important factors. To the authors’ knowledge, this is the first study investigating the potential of Rhodospirillaceae as a reservoir for resistance to antibiotics used in human medicine. The consequence for resistance prevalence in human pathogens and for their antibiotic therapy needs evaluation.