Veterinary and growth-promoting antibiotics are widely used in animal husbandry and accumulate in manure-fertilized soils. However, the impact of these antibiotics on symbiotic nitrogen fixation is poorly understood. We investigated the effect of the veterinary antibiotics oxytetracycline and sulfamethazine, and a combination of both, on nitrogen fixation in alfalfa (Medicago sativa L.) inoculated with Sinorhizobium meliloti. In a pot experiment, M. sativa was grown in soils fertilized with fresh manure that contained environmentally relevant antibiotic concentrations (0.2, 2, and 200 mg kg −1 ). Nodulation, nitrogen fixation, and nutrient concentrations were determined in the alfalfa plants and soils after 12 wk. Compared with the antibiotic-free control, symbiotic nitrogen fixation increased significantly in soils mixed with manure containing 2 and 200 mg kg −1 oxytetracycline (20.1 and 20.8% increase, respectively) and a mixture of 200 mg kg −1 oxytetracycline and sulfamethazine (12.4% increase). The measured plant-and soil-related parameters failed to explain the observed increase in nitrogen fixation. However, using concentration levels that accurately reflect common agricultural practices, we obtained results that directly contradict other experiments conducted under unrealistically high antibiotic concentrations.