The conversion of native habitats into anthropogenic ones compromises the original composition and configuration of the landscapes, influencing ecological dynamics and affecting biodiversity. Increasingly, landscape ecology has shown that these effects can only be understood if they are accessed at adequate spatial scales, as the scale at which landscape structure is evaluated influences species responses. Here, we investigated how three variables of landscape composition (proportion of forest, coffee crop, and pasture) and two of configuration (number of fragments and mean nearest neighbor distance) interfere with the richness and composition of medium- and large-sized mammals, considering a multiscale approach. We recorded medium- and large-sized mammal species in 13 landscapes with predominant matrices of coffee and pasture in Minas Gerais, Brazil. Then, we built distance-based linear models to identify the scale of effect of each landscape variable for both response variables considering eight scales (from 250 m to 2000 m). Finally, we verified the influence of the landscape on the richness and composition of mammals, considering the landscape variables in their respective scales of effect. We found 67% of the probable species occurring in the region. The scales of effect varied among landscape variables, probably due to the fact that different variables affect different aspects of organisms’ ecological requirements. The proportion of pasture in the landscape explained the variation in species richness, while the proportion of forest explained the variation in species composition. In addition, the proportion of pasture in the landscape had a positive influence on species richness, indicating that this matrix may favor the presence of generalist species of habitat and/or this result is due to the higher concentration of species in the fragments immersed in this matrix. These results suggest that considering different responses to biodiversity is important to understand different aspects of the landscape’s influence on biodiversity. In addition, the composition of the landscape is fundamental for the perpetuation of species and, therefore, both forest cover and types of matrices in the landscape must be considered to improve species conservation strategies. Finally, generalizing a spatial scale can lead to misinterpretations about the influence of the landscape on biodiversity.