<p>The possibility of environmental contamination associated with chloramphenicol requires special attention. The primary challenges in the biodegradation of chloramphenicol from the contaminated environment are obtaining effective antibiotic-degrading microbes of pure cultures and determining the entire metabolic pathway involved in efficient degradation. The potent strain <i>Aspergillus neoflavipes</i> AJC3 was able to degrade 98.70% of chloramphenicol at a concentration of 50&#xa0;mg/L within 8&#xa0;d. The biodegradation pattern of chloramphenicol was evaluated by employing high-performance liquid chromatography (HPLC) and validated through gas chromatography mass spectroscopy (GC–MS), liquid chromatography mass spectrometry (LC–MS) and Fourier transform infrared spectroscopy (FTIR). Three metabolic compounds were identified during the chloramphenicol biodegradation process and the degradation pathway involved zero-order and first-order kinetics model according to the kinetics energy. And to evaluate the effects of chloramphenicol in aquatic environment, toxicity studies were conducted using the nauplii and cysts of <i>Artemia salina</i> and the nauplii mortality was evaluated at 24&#xa0;h.</p> Graphical abstract <p></p>