Context. Nest boxes provide important nesting, denning and shelter sites for many fauna species worldwide, but we know little about the factors that influence the suitability of nest boxes for particular species. Such information is crucial in urban landscapes where natural hollows are scarce. Aims. The aim of this study was to record the use of nest boxes by sugar gliders (Petaurus breviceps), squirrel gliders (P. norfolcensis) and other fauna in a peri-urban landscape in northern Victoria and examine factors at multiple spatial scales that may influence nest-box use. Methods. We monitored the use of 102 nest boxes over three seasons in 2006. Attributes that may influence nest-box occupancy were measured at five different spatial scales: (i) landscape; (ii) the habitat beyond 20 m of the nest box; (iii) the habitat within 20 m of the nest box; (iv) the tree that the nest box was located in; and (v) the nest box. Key results. At the landscape scale, topography influenced nest-box occupancy with squirrel gliders using boxes in. at or gully areas, and sugar gliders using boxes in gully, mid-slope or ridge areas. For habitat beyond 20 m of the nest box, sugar gliders were more likely to occupy boxes with a higher density of surrounding nest boxes and a higher density of residential dwellings. Within 20 m of the nest box, boxes occupied by sugar gliders were more likely to occur in areas with a higher density of acacia shrubs and lower density of hollow-bearing trees, whereas the presence of acacia did not influence nest-box use by squirrel gliders. At the scale of the nest-box tree, boxes occupied by sugar gliders were more likely to be on smaller trees (based on height and diameter) and on box (e.g. red box Eucalyptus polyanthemos) species. The only nest-box characteristic to have a strong relationship with occupancy was date of establishment, with longer established boxes more likely to be occupied. Conclusions. Our study demonstrates that various factors influence nest-box use at different scales and nest boxes remain an important conservation and management tool in heavily modified landscapes. Implications. Land managers and groups should be aware that nest boxes may help to alleviate some of the negative impacts of the loss of hollow-bearing trees in low density urban areas, but nest-box use will vary depending on landscape context, habitat factors, box design, and the ecological traits of the target species. Each of these factors must be considered to maximise the conservation benefits of nest-box programs.