Antibiotic residues in animal waste from concentrated animal feeding operations are of considerable concern because of the potential development of antibiotic resistant bacteria in the environment and the effect of these residues on manure treatment systems. The objective of this study was to determine the effect of cefazolin (10 mg L -1 ) during thermophilic and mesophilic anaerobic co-digestion of manure and waste milk from cows treated with cefazolin for mastitis. The collected antibiotic free manure samples were mixed with slurry and milk obtained from a healthy cow accordingly to form manure mixture (slurry 50 % and manure 50 %) and milk mixture (slurry 50 %, manure 45 % and milk 5 %), each of which was further separated as the control and cefazolin spiked (10 mg L -1 ), loaded into triplicate 1 L batch digesters and anaerobically digested at 37 °C and 55 °C for 22 days separately. Control and cefazolin spiked milk mixtures always produced significantly (p < 0.05) higher total and methane gas yields compared to the respective manure mixtures. Compared to the control in both digester types, no significant (p > 0.05) differences in total and methane gas yields were observed in the respective cefazolin spiked digesters despite the temperature at which the digestion was carried out. Volatile fatty acid (VFA) accumulation and reduction of pH were not observed in any digester of both substrates at both temperatures at the end of the experiment, which confirmed the process stability. However, further investigations are necessary to analyse the effects of increased concentrations of cefazolin in the substrates on digester stability.