The production of striped catfish (Pangasianodon hypothalamus) has increased worldwide; recently, it was farmed with Nile tilapia in polyculture farms. Polyculture systems and water temperature (25℃ and 33℃) could affect Edwardsiella tarda infection, antibiotic efficacy, and residues. Moribund fishes were collected from three Farms 1-3: Farm 1 (monoculture, Nile tilapia), Farm 2 (monoculture, striped catfish), and Farm 3 (polyculture). Four E. tarda, LAMSH1, and LAMAH2-4 were isolated, whereas LAMAH3 was isolated from both fish spp., where striped catfish were highly susceptible to infection. The obtained E. tarda, which was isolated from striped catfish, has a significantly lower LD(50) than those retrieved from Nile tilapia, and co-infection occurred only in striped catfish on Farm 3. The infection was screened and confirmed by gyrB1 gene presence while detecting the cds1, pvsA, and qseC genes indicated virulence. All isolates were sensitive to ciprofloxacin and florfenicol but showed resistance to a high number of other antibiotics, resulting in high multi-drug resistant (MDR) indices exceeding 0.2, except for strain LAMAH4, which had an index of 0.18.Analyses of farms water revealed high ammonia compounds total ammonia nitrogen (TAN), unionized ammonia (NH(3)), nitrite (NO(2)), and nitrate (NO(3)) in Farm 2 (monoculture, striped catfish), and the recorded significantly higher concentrations were 2.75, 0.29, 0.24, and 2.01 mg/L, respectively, which were compared with Farm 1 and Farm 3. In the indoor experiment, at high water temperatures (33 °C), Nile tilapia and striped catfish had a high mortality rate and re-isolation of E. tarda (10-20%) compared to those exposed to low water temperatures (25 °C). These observations were concurrent with low antibiotic residues in their hepatic tissues. Despite water temperature, Nile tilapia showed higher ciprofloxacin residues than striped catfish.The study concluded that striped catfish are more susceptible to the bacteria E. tarda compared to Nile tilapia, particularly in polyculture farms, which resulted in a higher infection rate. Both Nile tilapia and striped catfish exposed to elevated water temperatures exhibited increased vulnerability to bacterial infections. Additionally, these fish showed a high re-isolation rate of E. tarda while having low ciprofloxacin residues in their hepatic tissues.