Freshwater aquaculture is threatened by Aeromonas hydrophila, whose multidrug resistance limits the efficacy of antibiotics. Here, we investigated the antibacterial mechanisms of magnolol, a bioactive compound from Houpoea officinalis. Magnolol strongly inhibited A. hydrophila growth, impaired motility by affecting flagellar function, and disrupted membrane integrity. Proteomic analysis at a sublethal concentration revealed extensive remodeling of bacterial protein expression, with the sodium/glycine symporter GlyP markedly upregulated, suggesting disruption of glycine homeostasis. GO enrichment indicated enrichment of membrane-associated proteins and activation of envelope stress responses. Gene-deletion mutants further identified NagR, a GntR-family regulator of N-acetylglucosamine metabolism, as a potential target. ∆nagR grew better under magnolol stress, while molecular docking confirmed stable hydrogen bond interactions with NagR. Together, magnolol inhibits growth, motility, and membrane stability, while targeting NagR and disturbing metabolic homeostasis. These findings elucidate magnolol’s multifaceted antibacterial mechanisms and highlight its potential as a natural antimicrobial for aquaculture pathogen control and food safety improvement.