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In the Brazilian Amazon, private land accounts for the majority of remaining native vegetation. Understanding how land‐use change affects the composition and distribution of biodiversity in farmlands is critical for improving conservation strategies in the face of rapid agricultural expansion. Working across an area exceeding 3 million ha in the southwestern state of Rondônia, we assessed how the extent and configuration of remnant forest in replicate 10,000‐ha landscapes has affected the occurrence of a suite of Amazonian mammals and birds. In each of 31 landscapes, we used field sampling and semistructured interviews with landowners to determine the presence of 28 large and medium sized mammals and birds, as well as a further 7 understory birds. We then combined results of field surveys and interviews with a probabilistic model of deforestation. We found strong evidence for a threshold response of sampled biodiversity to landscape level forest cover; landscapes with