Nocturnal fluxes may be a significant factor in the annual water budget of forested ecosystems. Here, we assessed sap flow in two co-occurring evergreen species (Eucalyptus parramattensis and Angophora bakeri) in a temperate woodland for 2 years in order to quantify the magnitude of seasonal nocturnal sap flow (En) under different environmental conditions. The two species showed different diurnal water relations, demonstrated by different diurnal curves of stomatal conductance, sap flow and leaf water potential. The relative influence of several microclimatic variables, including wind speed (U), vapour pressure deficit (D), the product of U and D (UD) and soil moisture content, were quantified. D exerted the strongest influence on En (r² = 0.59–0.86), soil moisture content influenced En when D was constant, but U and UD did not generally influence En. In both species, cuticular conductance (Gc) was a small proportion of total leaf conductance (Gs) and was not a major pathway for En. We found that En was primarily a function of transpiration from the canopy rather than refilling of stem storage, with canopy transpiration accounting for 50–70% of nocturnal flows. Mean En was 6–8% of the 24-h flux across seasons (spring, summer and winter), but was up to 19% of the 24-h flux on some days in both species. Despite different daytime strategies in water use of the two species, both species demonstrated low night-time water loss, suggesting similar controls on water loss at night. In order to account for the impact of En on pre-dawn leaf water potential arising from the influence of disequilibria between root zone and leaf water potential, we also developed a simple model to more accurately predict soil water potential (ψs).