Nocturnal water flux has been observed in trees under a variety of environmental conditions and can be a significant contributor to diel canopy water flux. Elevated atmospheric CO₂ (elevated [CO₂]) can have an important effect on day-time plant water fluxes, but it is not known whether it also affects nocturnal water fluxes. We examined the effects of elevated [CO₂] on nocturnal water flux of field-grown Eucalyptus saligna trees using sap flux through the tree stem expressed on a sapwood area (Js) and leaf area (Et) basis. After 19 months growth under well-watered conditions, drought was imposed by withholding water for 5 months in the summer, ending with a rain event that restored soil moisture. Reductions in Js and Et were observed during the severe drought period in the dry treatment under elevated [CO₂], but not during moderate- and post-drought periods. Elevated [CO₂] affected night-time sap flux density which included the stem recharge period, called 'total night flux' (19:00 to 05:00, Js,r), but not during the post-recharge period, which primarily consisted of canopy transpiration (23:00 to 05:00, Js,c). Elevated [CO₂] wet (EW) trees exhibited higher Js,r than ambient [CO₂] wet trees (AW) indicating greater water flux in elevated [CO₂] under well-watered conditions. However, under drought conditions, elevated [CO₂] dry (ED) trees exhibited significantly lower Js,r than ambient [CO₂] dry trees (AD), indicating less water flux during stem recharge under elevated [CO₂]. Js,c did not differ between ambient and elevated [CO₂]. Vapour pressure deficit (D) was clearly the major influence on night-time sap flux. D was positively correlated with Js,r and had its greatest impact on Js,r at high D in ambient [CO₂]. Our results suggest that elevated [CO₂] may reduce night-time water flux in E. saligna when soil water content is low and D is high. While elevated [CO₂] affected Js,r, it did not affect day-time water flux in wet soil, suggesting that the responses of Js,r to environmental factors cannot be directly inferred from day-time patterns. Changes in J s,r are likely to influence pre-dawn leaf water potential, and plant responses to water stress. Nocturnal fluxes are clearly important for predicting effects of climate change on forest physiology and hydrology.