The use of manure as a fertilizer is a common agricultural practice that can improve soil physicochemical and biological properties. However, antibiotics and their metabolites are often present, leading to the adaptation of soil bacterial communities to their presence. The aim of this study was to assess the effects of the extensively used, broad-spectrum antibiotic oxytetracycline on soil microbial community adaptation using a pollution-induced community tolerance assay. Manure-amended soil was spiked with oxytetracycline (0, 2, 20, 60, 150, and 500 mg kg−1) three times every ten days in the selection phase. The detection phase was conducted in Biolog EcoPlates with a second oxytetracycline exposure (0, 5, 20, 40, 60, and 100 mg L−1). All treatments demonstrated decreased metabolic activity after exposure to ≥ 5 mg L−1 oxytetracycline during the detection phase. Meanwhile, a significant increase in tolerance was observed following exposure to ≥ 20 mg oxytetracycline per kg soil during the selection phase. Therefore, the pollution-induced community tolerance approach with Biolog EcoPlates was a useful system for the detection of antibiotic selection pressures on soil bacterial communities. It is important to properly manage animal waste before their application to the soil to reduce the occurrence of antibiotic-resistance in the environment.