The role of private land in biodiversity conservation is recognized, yet has received limited attention in the literature. Increasing urban development on the rural–urban interface of regional cities in Australia is creating a fragmented landscape of urban and conservation areas. Residents in these areas can assist in biodiversity conservation. Data on residents’ pro-conservation behaviours, knowledge, attitudes and connection to nature were collected using a mail questionnaire survey with 300 returned. Drawing on a range of theoretical frameworks, relationships between environmental knowledge, connection to nature and pro-environmental behaviours were analysed. Findings revealed that many residents participate in a variety of pro-conservation behaviours and are supportive of biodiversity. However, their knowledge of local biodiversity is low and are less likely to participate in community-based conservation actions. Study findings can assist local government and conservation organisations tailor their biodiversity conservation strategies to enhance biodiversity conservation on private land.