<p>Groundwater nitrate contamination is a critical environmental challenge, demanding sustainable, low-energy treatment technologies. Although microalgal biofilm photobioreactors (PBRs) are promising for nitrate removal, the influence of hydraulic retention time (HRT) on biofilm structure and treatment performance remains unclear. In this study, a mixed <i>Chlorella vulgaris</i>–<i>Scenedesmus</i> sp. biofilm was developed on the inner walls of a vertical bubble-column PBR to treat nitrate-contaminated groundwater (50 mg NO₃⁻–N L^-1). Biofilm formation was initiated under batch operation and transitioned to continuous up-flow operation with stepwise reductions in HRT from 6 days to 14 hours. NO₃⁻–N, zeta (ζ)-potential, optical density (OD₇₅₀), and biofilm thickness were monitored to assess biofilm development, stability, and its performance. During batch operation, NO₃⁻–N dropped below 10 mg L^-1 within 6 days, accompanied by a ζ-potential shift from −24 to −5 mV, indicating enhanced microalgal attachment, and an OD₇₅₀ increase from 0.08 to 0.3, reflecting biomass transition from suspension to surface-attached biofilm. Under continuous operation, achieved an NO₃⁻–N concentration of 11 mg L^-1, a 60 µm biofilm, and −10 to −5 mV ζ-potential. Reducing HRT to 4 days slightly decreased biofilm thickness (59 µm) and caused variable NO₃⁻–N (10–25 mg L^-1). At HRTs ≤2 days, biofilms compacted (57 µm), partially detached, and demonstrated higher NO₃⁻–N (20–28 mg L^-1), despite ζ-potential approaching −3 mV. Consequently, HRT controls biofilm thickness and structure for nutrient penetration and stability, while ζ-potential and OD₇₅₀ variations serve as reliable indicators, offering practical guidance for optimizing microalgal biofilm PBRs in groundwater nitrate remediation.</p> Graphical abstract <p></p>