<p>Bronopol (C₃H₆BrNO₄) is newly used as an antimicrobial in aquaculture. Our study investigates the time-dependent effects of the antimicrobial Bronopol during <i>Aeromonas hydrophila</i> infection on Nile tilapia (<i>Oreochromis niloticus</i>). Fish were divided into four groups: a control (G0), Bronopol (0.333&#xa0;µl/L) water additives (G1), <i>A. hydrophila</i>-injected (G2), and the combined <i>A. hydrophila</i>-injected and Bronopol-added (G3) for 7 days. G2 generally led to significant (<i>P</i> &lt; 0.05) decreases in RBCs, Hb, PCV, monocytes, lymphocytes, and digestive enzyme activities (amylase, lipase, and protease), alongside significant (<i>P</i> &lt; 0.05) increases in heterophils, liver enzymes (AST, ALT, ALP), kidney markers (creatinine, urea), and indicators of oxidative stress (MDA). G1 often induced an initial inflammatory response (increased WBCs and RBCs at 6&#xa0;h) and higher (<i>P</i> &lt; 0.05) levels of immune parameters (lysozyme, phagocytic activity, and IgM). G3 exhibited some mitigation of pathological effects in histopathology compared to G2 but also showed significantly (<i>P</i> &lt; 0.05) higher NBT reduction and distinct gene expression patterns. Gene expression analysis revealed tissue- and time-specific changes; notably, G3 showed significant upregulation (<i>P</i> &lt; 0.05) of <i>hsp70</i>, <i>cox-2</i>, <i>il-1β</i>, and <i>tnf-α</i> in the liver at 6&#xa0;h, but downregulation in the kidney. Conversely, in 7 days, all gene expressions were significantly higher (<i>P</i> &lt; 0.05) in the G3 group in the liver, while in the kidney, they were lower in the same group. The study explores Bronopol’s efficacy in enhancing fish immunity to fight <i>A. hydrophila</i>, dependent on exposure timing and dose (0.333&#xa0;µl/L), necessitating careful management in aquaculture practices.</p> Graphical abstract <p></p>