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The maintenance of spontaneous weed cover is a conservation practice used in olive groves. Herbaceous plants in alleys between the trees can increase the capacity of this agroecosystem to remove carbon. However, the influence of this practice on carbon assimilation at the leaf scale has not yet been studied in olive trees. Also, the presence of other species competing with olive trees for soil water has the potential to modify the water use efficiency, a key parameter in a climate change context. In this study, leaf-scale net carbon assimilation (Aleaf), transpiration (Eleaf) and water use efficiency as the ratio Aleaf/Eleaf(WUEleaf) were quantified in olive grove divided by two different treatments: (1) a weed-free (WF) ecosystem in which weed growth was inhibited by applying herbicide; and (2) a weed-covered (WC) ecosystem in which spontaneous herbaceous plants were kept and then mechanically mowed and left on the ground. A portable leaf photosynthesis system was used to measure olive leaf fluxes for both treatments, and likewise for the ecosystem scale via two eddy covariance towers assessing gross primary production (GPPeco), evapotranspiration (ETeco), and water use efficiency (WUEeco). We found that the average Aleaf was 24% higher in the WF treatment while GPPeco decreased 32% compared to WC treatment. However, Aleaf was significantly different between treatments only during weed growth: January-May (Aleaf-WF = 7.6±3.7 μmol CO2m−2s−1; Aleaf-WC = 5.1±3.1 μmol CO2m−2s−1) while Aleaf was similar between the two treatments after mowing. Mowed weeds decreased Tsoil and VPD, and these changes were accompanied by a decrease in Eleaf in olive trees. Therefore, this led to WUEleaf-WF>WUEleaf-WC when the weeds were growing and the opposite after mowing. Thus, although the presence of spontaneous weeds increased the annual ecosystem C uptake in the olive orchard, both Aleaf and seasonal fluctuations in WUEleaf were reduced with weed maintenance.