Isolated trees are increasingly recognised as playing a vital role in supporting biodiversity in agricultural landscapes, yet their occurrence has declined substantially in recent decades. Most bats in Europe are tree-dependent species that rely on woody elements in order to persist in farmlands. However, isolated trees are rarely considered in conservation programs and landscape planning. Further investigations are therefore urgently required to identify which trees – based on both their intrinsic characteristics and their location in the landscape – are particularly important for bats. We acoustically surveyed 57 isolated trees for bats to determine the relative and interactive effects of size, tree-related microhabitat (TreM) diversity and surrounding landscape context on bat activity. Tall trees with large diameter at breast height and crown area positively influenced the activity of Pipistrellus pipistrellus and small Myotis bats (Myotis spp.) while smaller and thinner trees favoured M. myotis activity. The diversity of TreMs that can be used as roosts had a positive effect on (i) Barbastella barbastellus activity only when trees were relatively close ([removed]10% within 100 radius scale). The potential benefits of isolated trees for bats result from ecological mechanisms operating at both tree and landscape scales, underlining the crucial need for implementing a multi-scale approach in conservation programs. Maintaining the largest and most TreM-diversified trees located in the most heterogeneous agricultural landscapes will provide the greatest benefits.