Abstract: Microbially mediated denitrification is essential for nitrate removal in groundwater. Fluoroquinolones (FQs) pollution, mainly from reclaimed water and livestock farming, often co-occurs with nitrate and may influence denitrification. Recent studies have identified the effects of FQs on denitrification in groundwater systems under varying exposure patterns of FQs, microbial characteristics, and environmental conditions. The underlying mechanisms have been elucidated from multiple perspectives, including community structure, metabolic pathways, and molecular-level responses. (1) FQs exhibit concentration- and compound-specific effects. At low concentrations, they may act as signaling molecules that regulate metabolic pathways and reshape microbial communities, thereby indirectly affecting denitrification. High concentrations of FQs inhibit bacterial growth, resulting in reduced denitrification efficiency and a delayed response. Combined FQs effects can be rapidly assessed using the microbial biomass determination method. (2) Environmental-microbial interactions modulate FQs effects. Microbial responses follow a “sensitive–non-sensitive–sub-sensitive” succession pattern, driven by bacterial abundance and resistance, with carbon metabolism and community structure changes as major regulatory pathways. Future research should focus on three areas: (1) the mechanisms and recovery pathways of denitrification inhibition under different FQs input scenarios; (2) the occurrence forms of FQs in groundwater and their multilayered impacts on denitrification functions; and (3) the development of a multi-scale coupled “FQs–microbe–environment” model to identify thresholds of critical drivers, thereby providing theoretical support and technical solutions for groundwater nitrogen pollution control. The BRIEF REPORT is available for this paper at http://www.ykcs.ac.cn/en/article/doi/10.15898/j.ykcs.202505050114.