Abstract: Oxytetracycline, as a commonly used growth-promoting antibiotic in the livestock breeding industry, is prone to residue in the manure, which poses a potential threat to the reproduction of soil microorganisms and crop growth when returned to the field. Most previous studies have set high concentration gradients, resulting in their limited application. Based on this, this article proposes to study the impact of actual antibiotic residues on bacterial community diversity to provide a theoretical basis for the safe application of organic fertilizers. In this study, the addition of oxytetracycline was set at 0.2 mg/kg, and high-throughput sequencing technology was employed to analyze bacterial community composition, structural changes, and types of significant enriched species. The results showed that the addition of oxytetracycline increased the relative abundance of Firmicutes and Actinobacteria, with a range from 1.1% to 5.0%; it also reduced the relative abundance of Proteobacteria, Chloroflexi, and Bacteroidetes, with a range below 2.0%. Compared to the control group (CK), Thermoactinomycetaceae were significantly enriched in the oxytetracycline group (OCK). Over time, oxytetracycline changed the structure of the bacterial community. This study proved that 0.2 mg/kg oxytetracycline can alter the relative abundance of soil bacterial communities and cause differences in community structure, leading to significant enrichment of Thermoactinomycetaceae in soil. Therefore, this study can provide inspiration for finding microorganisms that are effective against antibiotic pollution and lay the foundation for the safe application of organic fertilizers.