One Health (OH) is an interdisciplinary approach aiming to achieve optimal health for humans, animals and their environments. Case reports and systematic reviews of success are emerging; however, discussion of barriers and enablers of cross-sectoral collaboration are rare. A four-phase mixed-method Delphi survey of Australian human and animal health practitioners and policymakers (n = 52) explored areas of consensus and disagreement over: (1) the operational definition of OH; (2) potential for cross-sectoral collaboration; and (3) key priorities for shaping the development of an OH response to significantly elevated zoonotic disease risk. Participants agreed OH is essential for effective infectious disease prevention and control, and on key priorities for outbreak responses, but disagreed over definitions and the relative priority of animal health and welfare and economic considerations. Strong support emerged among Australian experts for an OH approach. There was also recognition of the need to ensure cross-sectoral differences are addressed.