An important unsolved question in hydrology science is the consequence of the Eucalyptus afforestation on groundwater recharge. Here, we assessed recharge estimates before and after converting pasture cover to Eucalyptus plantation. Groundwater levels, recharge, rainfall (P) and actual evapotranspiration (ET) were evaluated from 2004 through 2016 over an outcrop area of the Guarani Aquifer System (GAS) in the southeastern Brazil. Recharge was estimated using the water-table fluctuation method. We observed a decrease of 100 mm/year in groundwater levels after land use changed from pasture to Eucalyptus. The average recharge decreased from 407 mm/year (27% of mean P) to 194 mm/year (13% of mean P) after land use change over the study area. We found a recharge decrease of 19% in 2012 and 58% in 2013 under similar annual rainfall in comparison with the long-term mean recharge (from 2004 to 2016). Our results indicated that the high values of ET observed in Eucalyptus plantation decreases water availability for recharge. Therefore, our findings are important to agencies for decision-making in water resources regarding the management of land cover and land use.