Land use intensification, by which habitat loss, habitat fragmentation and increased land ownership subdivision occurs, represents one of the largest threats to biodiversity. The extent to which land use intensification affects the presence of native mesocarnivores is largely unexplored, with great implications for all working landscapes where agriculture and native wildlife co-occur. We obtained mesocarnivore detection/non-detection data from 180 4-km2 sampling units in agricultural landscapes of southern Chile from January to April 2019. We used these data to (a) investigate the associations of private land ownership subdivision, forest fragmentation and forest loss with the occurrence of mesocarnivores using single-species occupancy models, (b) assess patterns of mesocarnivore co-occurrence with free-roaming domestic mesocarnivores (e.g. cats and dogs) using two-species occupancy models and (c) determine whether co-occurrence of native and domestic mesocarnivores led to alterations in species’ temporal activity. Land ownership subdivision, rather than habitat loss or fragmentation, had the greatest impact on native mesocarnivore occurrence, with some influence of domestic dogs. Mesocarnivore community occurrence varied from a native to domestic species composition as private land ownership subdivision increased. Native mesocarnivores altered their behaviour temporally when co-occurring with domestics. Lastly, the presence of domestic dogs was associated with an absence of native mesocarnivores, which merits further investigation into the contribution of domestic dogs to a defaunation process in agricultural areas. Policy implications. Our evidence supports focusing efforts in three key dimensions to advance biodiversity conservation in agricultural landscapes. First, private land subdivision represents a robust proxy for measuring anthropogenic impacts on mesocarnivores, and we advocate its use to inform agricultural policy to mitigate a potential defaunation process. Second, there is a need to further engage with landowners and evaluate values, motivation, willingness and action to protect remnant native vegetation and slow land use change. And, lastly, improvements to legislation and conservation marketing strategies on responsible pet ownership are critical to ameliorating the negative impacts of dogs on native wildlife species.