ABSTRACTRecently, nanoparticles have attracted attention as a preventive tool for certain infectious diseases affecting fish in aquaculture. Furthermore, freshwater fishes are frequently vulnerable to summer mass morality caused by Aeromonas bacteria. In this regard, we focused on the evaluation of the in vitro and in vivo antimicrobial activity of chitosan (CNPs) and silver (AgNPs) nanoparticles againstAeromonas hydrophila subsp. hydrophila. CNPs and AgNPs were prepared at a mean particle size of 9.03 and 12.8 nm and a charge equalled+36.4 and −19.3 mV for CNPs and AgNPs, respectively.A.hydrophila subsp. hydrophila, Aeromonas caviae, and Aeromonas punctatawere retrieved and identified by traditional and molecular techniques. The sensitivity of the obtained bacteria to eight different antibiotic discs was also tested. The antibiotic sensitivity studies revealed the presence of multidrug-resistant (MDR)Aeromonasspecies (spp.). The bacterium that showed the highest multidrug resistance against the tested antibiotic discs wasAeromonas hydrophila subsp. hydrophila. Therefore, CNPs and AgNPs were in vitro tested against the isolated bacterium and exhibited inhibition zones of 15 and 25 mm, respectively. TEM images also showed that CNPs and AgNPs had an antagonistic action against the same bacterium causing loss of architecture and bacterial death.