Abstract The objective of this study was to determine the effect of administration of oxytocin prior to milking on enzyme activities and thiocyanate concentration in bovine milk. Intramuscular administration of oxytocin (20 IU) was made to a group of cattle comprising 8 animals, whilst a second group was kept as control. Significant (p < 0.01) differences were measured in enzymatic levels of oxytocin-treated and control bovine milk. Alkaline phosphatase and lipase activity were reduced in milk of oxytocin-treated cows. However, the trend was the opposite for acid phosphatase, where high enzyme activity was observed in milk after oxytocin treatment (0.010 U mL−1) compared with the control (0.004 U mL−1). Treating cattle with oxytocin did not significantly (p < 0.01) affect lactoperoxidase concentration but increased thiocyanate levels of milk by ∼6.5%. In conclusion, oxytocin administration to cows cause variations in milk enzyme activities and might consequently affect milk stability against certain deteriorative changes.