The Hawaiian Archipelago stretches 2500 km from the Main to the Northwestern Hawaiian Islands, represents a complex gradient of oceanographic and anthropogenic drivers, and has a high abundance and diversity of native and invasive macroalgae. These photosynthetic organisms occur in intertidal to mesophotic (30–150+ m) depths and absorb nitrogen with limited fractionation associated with their physiology and source. Our goal was to examine nitrogen dynamics from shallow to mesophotic reefs using compositional patterns of two well‐characterized macroalgal tissue parameters: stable isotope ratio of nitrogen and tissue nitrogen content. We collected 813 macroalgal samples from 13 islands/atolls between 0 and 117 m depths. Within the Main Hawaiian Islands, macroalgal tissue stable N isotope ratios were higher in mesophotic depths; N content was higher in shallow depths. However, within the Northwestern Hawaiian Islands, no differences in stable N isotope ratios and N content were found between shallow and mesophotic depths. Regionally, stable N isotope ratios varied along a gradient of anthropogenic and oceanographic processes (in Main and Northwestern Hawaiian Islands, respectively), while N content reflected elevated nitrogen in the Main compared with the Northwestern Hawaiian Islands. Additionally, the invasive macroalga Avrainvillea lacerata had significantly higher N content than co‐occurring native bryopsidalean macroalgae at similar depths, and may be reshaping nutrient dynamics from shallow to mesophotic depths in the Main Hawaiian Islands. Nitrogen dynamics at mesophotic depths may be influenced by nearshore anthropogenically derived nitrogen via submarine groundwater discharge and/or inputs from deeper water within the Main Hawaiian Islands.