This dataset provides analysis results for recent status of and trends in 44 fish and 99 macroinvertebrate assemblages in the Chesapeake Bay watershed. For both biological datasets a multi-metric index, metric of assemblage sensitivity, functional feeding group metric and habitat preference metric was selected for analysis. Sites used to calculate trends required a minimum of 7 years of data. Status was calculated as a three-year average by averaging metric values for the most recent three years of data at each site and trends were calculated using generalized additive models (GAM) in the R package 'mgcv' for the full window of sample years at each site. Detailed data preparation information, analytical methods, and results are presented and discussed in the associated Scientific Investigative Report (https://doi.org/10.3133/sir20255072).