Excessive use of antibiotics potentially threatens human health, agricultural production, and soil phytoremediation. This arouses concern over the potential adverse effects of a commonly used antibiotic, oxytetracycline (OTC), on plants used for soil remediation and possible stimulation of antibiotic resistance genes in soils. A greenhouse experiment was conducted to investigate different rates (0, 1, 5, and 25 mg kg −1 ) and frequencies (one single high and daily low application) of OTC addition to soil on phytoremediation of a heavy metal contaminated soil by Sedum plumbizincicola and/or Medicago sativa (alfalfa). After 90 days both Cd and Zn were substantially removed by phytoextraction into S. plumbizincicola shoots especially at the high OTC (25 mg kg −1 ) treatment which also led to inhibition of antioxidative enzyme activities in both plant species. Soil microbial activity decreased significantly with the addition of OTC, and this was ameliorated by planting alfalfa and S. plumbizincicola together. OTC at <5 mg kg −1 increased the biomass of both plant species, but the frequency of OTC addition had no effect on the rate of metal removal. Alfalfa exhibited greater detoxification ability and effectiveness in soil microbial activity promotion than S. plumbizincicola with intercropping. Phytoremediation by alfalfa and S. plumbizincicola in association can both promote the removal of heavy metals and also alleviate the toxic effects of pollutants on plants and soil microbes even at relatively high soil OTC concentrations.