The purpose of this study was to estimate changes to aquatic habitat availability and fish and aquatic macroinvertebrate assemblages in coastal plain streams that may result from groundwater withdrawals. Hypothetical streamflow reductions of 5, 10, 20, and 30% of average annual streamflow and the 7-day low flow were calculated for 14 study sites. Using study-site streamflow and channel morphology data, relationships were developed to estimate five physical habitat metrics including average stream width, stream depth, stream cross-sectional area, stream-reach volume, and stream velocity. A second set of relationships was then developed to estimate the percentage reduction to each habitat metric under the hypothetical streamflow reduction scenarios. The average percentage decrease and variability of each habitat metric increased with successive streamflow reductions. Average percentage decreases in stream width, stream depth, stream cross-sectional area, and stream-reach volume were more pronounced for the reductions of average flow, whereas the average percentage decreases in stream velocity were more pronounced for the reductions of low flow. Models derived from a separate fish and macroinvertebrate study were used to estimate assemblage structure responses based on hypothetical reductions in average annual streamflow. The results of this study can be used to estimate the potential impact of groundwater withdrawals and subsequent streamflow reductions on available aquatic habitat and changes to fish and macroinvertebrate assemblages in coastal plain streams. Copyright © 2011 John Wiley & Sons, Ltd.