Plantation forestry is an increasingly important land use in Australia. While plantations can present significant environmental benefits such as salinity and erosion controls, their impact on streamflow needs to be recognized. This study evaluated the impacts of plantation and climate variability on streamflow from catchments in Australia. In total 15 catchments were selected based on the availability of streamflow, meteorological, and plantation information. The catchment areas range from 0.6 to 1136 km2 and represent different climatic conditions and management practices. The Mann–Kendall test was used to estimate trends in the annual streamflow, rainfall, potential evaporation, and runoff ratio. The plantation effect on streamflow was determined with the time-trend analysis method, while the effect of climate variability was calculated using the sensitivity-based approach. All the selected catchments showed reductions in annual streamflow and runoff ratio over the period of the records. The results indicate that plantation expansions accounted for 28% to 106% of the observed total streamflow reductions, while climate variability was responsible for 5% to 80% of the total streamflow reductions. It is evident that both plantation expansions and climate variability contributed to the observed streamflow reductions. In most of the catchments, the effect of plantation expansion on streamflow was more important than that of climate variability and is likely to have exacerbated the effect of climate variability on streamflow. Plantation expansions can reduce streamflow from both small and large catchments as this study demonstrated and the relative streamflow reduction can be linearly related to percentage plantation area in a catchment.