Highly altered agricultural watersheds like the Mississippi Alluvial Plain (MAP) in Mississippi, USA pose significant challenges for developing biological stressor-response relationships to monitor ecological improvements from best management practices (BMPs). Previous research (Taylor et al., 2023, https://doi.org/10.1016/j.ecolind.2023.110377) has indicated that MAP streams have elevated nutrient and ion concentrations as well as reduces biological diversity relative to other regions of MS. These data, collected for the Mississippi Department of Environmental Quality (MDEQ), are part of a joint effort to overcome these challenges and establish biological monitoring and assessment tools for MAP streams based on multiple biological assemblages including macroinvertebrates, diatoms, and bacteria (see DeVilbiss et al., 2023, https://doi.org/10.1111/gcb.16905). This data release contains macroinvertebrate, diatom, and stream habitat data collected in July-Sept 2021. Corresponding bacterial DNA sequencing data can be accessed at the National Center for Biotechnology Information's Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra) under accession number PRJNA909314. Stream water quality data can be accessed from the National Water Information System. Sediment texture and nutrient data analyzed at the USDA-ARS National Sedimentation Laboratory (Oxford, MS) can be accessed in the supplemental information of DeVilbiss et al., 2023 (https://doi.org/10.1111/gcb.16905).