Abstract Since European settlement, Eucalyptus box woodlands have been substantially modified by agricultural practices, and in many areas in southern Australia are now restricted to scattered or clumped trees. We report here on a study to examine the impact of trees on water flow (infiltration) in an agricultural landscape with substantial areas of extant native vegetation. We examined infiltration through coarse- and fine-textured soils within four landscape strata, the zones below Eucalyptus melliodora and Callitris glaucophylla canopies, the intertree zone dominated by perennial grasses and a landscape homogenized by cultivation and dominated by annual crops. We measured sorptivity, the early phase of water flow, and steady-state infiltration with disc permeameters at two supply potentials. These different potentials enabled us to separate infiltration into (i) flow through large (biopores) and small pores and (ii) flow through small pores only where biopores are prevented from conducting water. On the fine-textured soils, both sorptivity and steady-state infiltration were significantly greater (approximately fivefold) under the timbered strata compared with the grassy slopes or cultivation. Differences were attributable to the greater proportion of macropores below the tree canopies compared with the nontimbered strata. The lack of a significant difference on the coarse-textured soils, despite their macropore status, was attributed to differences in surface litter and plant cover, which would maintain continuous macropores at the surface and thus conduct large amounts of water. The tendency of slopes covered by cryptogamic crusts and grasses to shed run-off and for the trees to absorb substantial quantities of water reinforced the important ecological service provided by trees, which moderates large run-off events and captures small amounts of water leaking from the grassy patches. In the absence of these ‘ecosystem wicks’, run-off would find its way into regional groundwater and contribute to rising salinity.