The effects of time elapsed after milking and lactation stage were assessed on the lactoperoxidase (LP) enzyme activity and concentrations of thiocyanate in Sahiwal cattle (N = 15) and Nili‐Ravi buffaloes (N = 15). The LP activity and thiocyanate concentrations decreased as the time elapsed after milking (P < 0.05) but the concentration of these components increased as the lactation advanced. The LP enzyme activity was sufficient until 12 h post milking but the thiocyanate concentration after 8 h decreased to an insufficient level to activate the LP System, hence additional thiocyanate would be beneficial from external sources. No difference was noted regarding the LPS activity in cattle and buffaloes at the time of collection and subsequently after storage. However, at the end of lactation, the thiocyanate concentration was lower in buffaloes than that in cattle. (P < 0.05).