Possible adverse effects of a commonly used veterinary antibiotic, oxytetracycline (OTC), on acidogenic phase of anaerobic digestion of cattle manure along with optimum operating conditions were investigated. A standard veterinary practice of 50 ml OTC solution (20 mg/kg cattle weight) was injected into the muscles of cattle and then manure samples were collected for 5 days following the injection. The 5-day samples were equally mixed and used throughout digestion experiments. Preliminary batch tests were conducted to obtain the optimum pH range and observe volatile fatty acids (VFAs) production. In this regard, different sets of batch digesters were operated at pH ranging from 5.2 +/- 0.1 to 5.8 +/- 0.1 at mesophilic conditions with total solids content of 6.0 +/- 0.2%. The pH of 5.5 +/- 0.1 was found to be the optimum value for acidification for both non-medicated and OTC-medicated conditions. Under predetermined conditions, maximum total VFA (VFAtot) of 830 +/- 3 mg (as acetic acid)/L was produced and maximum acidification rate was evaluated as 11% for OTC-medicated cattle manure, whereas they were 900 +/- 6 mg (as acetic acid)/L and 12% for non-medicated manure. Digestion studies were further continued in a semi-continuous mode at pH 5.5 +/- 0.1 and SRT/HRT of 5 days. VFAtot concentrations and maximum acidification rate increased up to 2181 +/- 19 mg (as acetic acid)/L and 29% for non-medicated cattle manure. For OTC-medicated cattle manure, lower acidification rate of 18% was observed.