In Australian temperate woodlands, most squirrel glider (Petaurus norfolcensis) habitats exist outside formal conservation reserves, often in highly fragmented agricultural landscapes. To conserve squirrel glider populations in such woodlands it is essential to define important habitats and understand how they are used. This study examines the nocturnal habitat use of squirrel gliders across five sites within an agricultural landscape in south-eastern Australia. Over a five-month period we radio-tracked 32 gliders to 372 nocturnal locations. We quantify characteristics of key nocturnal habitats and describe their use. Gliders were more likely to use large eucalypt trees, particularly yellow box (Eucalyptus melliodora) and mugga ironbark (E. sideroxylon). Nocturnal activity mostly took place high in the canopy of eucalypts, accounting for 74% of fixes. Multiple regression models revealed that feeding was more likely to occur in large, healthy trees close to drainage lines, with a preference for E. melliodora, when eucalypts were not flowering. Flowering trees were preferentially sought and were strongly associated with being large healthy trees that occur on ridges and upper slopes. Showing that the squirrel glider utilises key feeding structures (large healthy Eucalyptus trees) in different parts of the landscape at different times has direct management implications in the conservation and restoration of squirrel glider habitat, particularly in fragmented temperate woodland.