Background and aims <p>Animal manure is a valuable fertilizer, and its proper use is essential in circular agriculture. However, antibiotics are commonly administered to livestock and excreted in manure, thereby entering soil ecosystems. The effects of antibiotic-containing manure on soil nitrogen (N) cycling microbial guilds, plant productivity, and N turnover in grassland ecosystems remain unclear.</p> Methods <p>In a two-factorial greenhouse pot experiment, we evaluated the impact of manure with different antibiotic residues in four plant communities: grass monoculture, clover monoculture, grass-clover culture, and a no-plant control. The fertilization treatments included antibiotic-free manure, manure containing oxytetracycline, and manure containing sulfadiazine. We measured soil N-cycling functional genes, aboveground and belowground plant biomass, clover symbiotic N fixation, soil mineral N pools, N₂O emissions, and antibiotic residues in plants and soil.</p> Results <p>Oxytetracycline, but not sulfadiazine, significantly increased the relative abundance of ammonia-oxidizing archaea (AOA) and marginally increased the abundance of N-fixing microbes across all plant communities. In clover monoculture, both antibiotics reduced root biomass and root total N content. However, antibiotic residues in soil did not have significant impacts on N fixation of clover, soil mineral N pools, and soil N₂O emissions.</p> Conclusion <p>At environmentally relevant concentrations, oxytetracycline residues in manure-amended soils could change the soil microbial community composition, favoring more tolerant or resistant groups such as AOA. Clover exhibited greater sensitivity to antibiotic exposure than grass. Further research is necessary to understand the long-term ecological consequences of persistent antibiotics like oxytetracycline in grasslands.</p>