. 1. We analysed the canopy and understorey communities of flies in the family Tachinidae, the most diverse group of parasitoid Diptera, in a small and isolated temperate plain forest in northern Italy. Our objective was to assess whether and how these communities differ from one another, and how species distribution relates to forest structure, host distribution, mating sites, and season. 2. The study was carried out in 2008 with 14 Malaise traps installed between April and November in an equal number of sites randomly selected inside the forest, seven on the ground and seven in the tree canopy. 3. Overall species richness, abundance, and turnover were greater in the understorey traps, but most diversity metrics indicate greater overall diversity and evenness in the canopy traps. Community ordination and estimates of beta diversity indicate that the two habitat-associated communities are distinct and should both be considered in assessments of insect diversity and community structure. Indicator species values revealed the presence of a number of species that were effective indicators of canopy and understorey habitats. No strong male bias in canopy traps was observed across species; however, the only significant sex ratio biases in the canopy were towards males. Both male and female biases were observed in understorey traps, depending upon the species.