Objective <p><i>Aeromonas hydrophila</i>, a pathogen of concern in aquaculture, utilizes quorum sensing mechanism to form biofilms that significantly increase the risk of infection in aquaculture commodity such as Nile tilapia (<i>Oreochromis niloticus</i>). The biofilm structure produced by <i>A. hydrophila</i> not only enhances its resistance to antibiotics but also complicates treatment efforts.</p> Results <p>In this study, we investigated the anti-quorum-sensing and biofilm disruption activities of bacterial extracts from <i>Streptomyces labedae</i> 20PM against <i>A. hydrophila</i> (in vitro and in vivo). Quantitative analysis showed that the crude extract had stronger anti-quorum-sensing activity in biofilms than the supernatant. Toxicity assessments indicated that both the supernatant and crude extract were non-toxic (24-hour LC₅₀ &gt;1000 ppm). GC-MS analysis identified 2-pentylpiperidine and (S)-2-propylpiperidine as the dominant compounds in the supernatant and crude extract, respectively. In vivo assays, including blood analyses and survival confirmed that incorporating the crude extract and supernatant into fish feed significantly contributed to the prevention and treatment of <i>A. hydrophila</i> infections in Nile tilapia. These findings suggest that the bioactive compounds derived from <i>S. labedae</i> 20PM have strong potential as natural agents for controlling <i>A. hydrophila</i> biofilm formation in aquaculture systems, offering a novel approach to mitigating bacterial infections.</p>