Our study aimed to investigate seasonal variation in the activity of arboreal and semi-arboreal mammals and investigate their overlap in temporal activity, as well temporal shifts in activity because of behavioural interference. In our camera trapping study in a fragmented landscape in south-eastern Australia, a total of ten arboreal and semi-arboreal species were found, with 35,671 independent observations recorded over 6517 camera trap nights. All species were found to be nocturnal; however, a notable number of daytime observations were made for several species (i.e. brown antechinus, Antechinus stuartii; sugar glider, Petaurus breviceps; bush rat, Rattus fuscipes; brown rat, Rattus norvegicus). Seasonal variations in diel activity were observed through an increase in crepuscular activity in spring and summer for antechinus, sugar gliders, brown rats, brushtail possums, Trichosurus vulpecula and ringtail possums, Pseudocheirus peregrinus. Diel activity overlap between species was high, that is 26/28 species comparisons had overlap coefficients (Δ) > 0.75. The species pair with the least amount of overlap was between southern bobucks, Trichosurus cunninghami and brown antechinus (Δ4 = 0.66). The species pair with the most overlap was between the native sugar glider and introduced brown rat (Δ4 = 0.93). When comparing the activity of sugar gliders in sites with low and high abundance of brown rats, sugar gliders appear to shift their activity relative to the brown rats. Similarly, behavioural interference was also observed between antechinus and sugar gliders, and when comparing sites of low and high abundance of sugar glider, antechinus had a shift in activity. Our work provides some of the first quantification of temporal patterns for several of the species in this study, and the first for a community of arboreal and semi-arboreal mammals. Our results indicate that some shifts in behaviour are potentially occurring in response to behavioural interference, allowing for coexistence by means of temporal partitioning.