The hydrologic regime of rivers and streams is a major determinant of habitat quality for fish and aquatic invertebrates. Long-term streamflow data were compiled and multidecadal streamflow trends and ecological flow (EFlow) statistics were calculated in support of the United States Geological Survey (USGS) Chesapeake Bay Science Initiative toward understanding fish habitat and health in the Chesapeake Bay Watershed (CBWS). A dataset comprising all streamgages (n = 409) reporting daily means of streamflow within the CBWS and remaining active as of September 30, 2018 (the end of Water Year [WY] 2018), independent of streamgage installation date, was retrieved from the USGS National Water Information System (NWIS). This dataset was then subset to include only those streamgages with a contiguous timeseries of streamflow data from a start date no earlier than April 1, 1939 (Climate Year [CY] 1940) and no later than October 1, 1999 (WY 2000). The R packages “EGRET” and "Eflowstats" were utilized together to determine streamflow trends and EFlow statistics from the subset (n = 243). Trends and EFlows were computed for the ranges 1940-1969 (n = 90), 1970-1999 (n = 167), and 2000-2018 (n = 243). Streamflow trends were computed for eight annual metrics (1-, 7- and 30-day minima [CY] and maxima [WY], mean and median [WYs]). These streamflow trends provide context for the 178 EFlow statistics (WY) which have been designated to characterize the magnitude, frequency, and duration of extreme high and low flows, the timing of seasonal flows, and the consistency of the historic regime. Files herein include the following Child Items: (1) a table summarizing streamflow trends for three time periods at a minimum of 90 and maximum of 243 streamgages and 500 time-series plots graphically representing those trends; (2) a table summarizing EFlow statistics and the change between each statistic for three time periods at a minimum of 90 and maximum of 243 streamgages; and (3) a GIS shapefile of the original 409 USGS streamgage locations, complete with NWIS attributes, active within the CBWS through September 30, 2018.