Promoting conservation practices in agriculture to protect biodiversity of rare Mediterranean ecosystems is nowhere more critical than in Chile, where less than 2% of the Mediterranean region is formally protected. We used the theory of planned behaviour to assess what influences Chilean winegrowers’ conservation behaviour and tested whether a sustainability programme was effective. We compared winegrowers involved in the programme with a comparison group, using semi-structured interviews at 23 wineries to determine predictors of conservation practice adoption at vineyards. The intervention group had higher levels of conservation behaviour than the comparison group and practised integrated pest management and exotic species control more frequently. Managers’ views on conservation practices as doing ‘what is right’ with regards to nature and the environment were evident in both groups. However, programme winegrowers recognized more cultural benefits of nature and reported a broader spectrum of organizational and community stakeholder influence. Economic resources were perceived as a major barrier, as well as the lack of data connecting biodiversity conservation with wine quality and production. This study demonstrates the multidimensional nature of winegrowers’ motivations and barriers for adopting conservation practices, which is critical to addressing the significant challenges facing biodiversity conservation and the promotion of sustainable agricultural systems.