<p>The impact of antibiotics on microbial-mediated denitrification has attracted widespread attention. The varying pH values of groundwater cause various antibiotics ionic forms, however, less attention on denitrification gets insufficient attention. Here, norfloxacin (NOR) was selected as a representative fluoroquinolone antibiotic to explore its differential effects on denitrification when it primarily exists in cationic (NOR⁺), zwitterionic (NOR^±), and anionic (NOR⁻) forms. The results showed that the NOR⁻ exerted the greatest inhibitory effect on nitrate reduction, with highest inhibition rates of 99.33%. Next was NOR^±, with inhibition rate of 55.12%. However, the NOR⁺ exhibited no impact. The inhibition of denitrification is the synergistic effects of microbial community, denitrification functional genes (DFGs) and antibiotic resistance genes (ARGs). NOR^± and NOR⁻ decreased the relative abundance of <i>Pseudomonas</i> and <i>Diaphorobacter</i> (<i>nirS</i>-type). While <i>Achromobacter</i> (<i>nirK</i>-type) and <i>Delftia</i> increased, indicating they might be potential antibiotic-resistant bacterial genera. Furthermore, NOR⁻ inhibited denitrification by suppressing <i>napA</i>, <i>narG</i>, and <i>nirS</i>, with inhibition rates of 99.74%, 99.52% and 99.46%, respectively. Meanwhile, NOR^± showed inhibition rates of 80.32% and 90.32% for <i>narG</i> and <i>nirS</i>. Notably, the inhibition rates tended to recover with prolonged NOR exposure. Additionally, ARGs analysis revealed distinct responses to NOR addition. The addition of NOR⁻ increased the relative abundance of 12 ARGs and led to the production of mobile genetic elements (MGEs). NOR^± induced 5 ARGs, while NOR⁺ did not exhibit a notable impact on ARGs. This study revealed the microbial mechanism behind the impact of charged antibiotic forms on denitrification in groundwater.</p> Graphical Abstract <p></p>