We have documented the antimicrobial activity of pouch secretions of the tammar wallaby, Macropus eugenii, over the period leading up to birth and after birth of the young animal. This activity was greatest against the Gram-negative Escherichia coli and highest at the time of birth. Fractionation of the pouch secretions showed that activity at different times over the reproductive periods was associated with different molecular-weight (MW) components, with compounds in the range up to 50 kDa active immediately prior to and at the time of birth. Proteomic analysis using 1 and 2DE PAGE and LC–MS/MS identified the major components of the pouch secretions at these times, at a range of pI's and MWs. The majority of high-confidence identifications, at a wide range of pI's and MW, were β-lactoglobulin, a known component of marsupial milk. We subsequently conducted a proteomic analysis of mammary gland secretions and digest products from the gut of the young animal, using 2DE PAGE and MALDI MS/MS, to confirm its source and compare it with the observed MW and pI's of β-lactoglobulin. Although we did not directly identify an effector molecule responsible for antimicrobial activity, these results lead us to propose that β-lactoglobulin plays a role in the protection of the young marsupial, a role previously thought to be primarily due to specific secretions from the epithelial surface of the pouch.