Oxytetracycline (OTC) which is a broad-spectrum veterinary tetracycline antibiotic is extensively used in poultry farms as a prophylactic, therapeutic, and growth stimulator. Upon administration, unmetabolized OTC is excreted from the animal body through droppings and accumulated in litter in the poultry industry. This study aimed at investigating the OTC degradation potential of an-OTC tolerant bacterial strain, isolated from poultry manure. The isolated strain’s morphology, biochemical properties, and 16S ribosomal RNA (rRNA) gene sequence confirmed that it belonged to the Lysinibacillus genus. To measure the residual OTC concentration, a high-performance liquid chromatography method was used. OTC degradation rates were 2.579 mg L−1d−1 with Lysinibacillus strain 3+I and 1.149 mg L−1d−1 without Lysinibacillus strain 3+I. In the presence of strain 3+I, the half-life significantly reduced to 2.68 days, compared to 6.03 days without strain 3+I. The strain demonstrated 85% removal with the OTC concentration of 10 μg/ml. The influence of pH, temperature, carbon sources, and nitrogen source, which influence degradation, were also investigated. The optimum condition favouring degradation was pH 6 at a temperature of 30°C. In addition, Lysinibacillus sp. strain 3+I’s ability to degrade OTC in poultry litter offers a promising approach to treat poultry manure and effluent containing OTC, preventing its contamination in the environment.