The swamp eel (Monopterus albus) is an important commercial farmed fish species in China. However, it is susceptible to Aeromonas hydrophila infections, resulting in high mortality and considerable economic loss. Povidone-iodine (PVP-I) is a widely used chemical disinfectant in aquaculture, which can decrease the occurrence of diseases and improve the survival. However, environmental organic matter could affect the bactericidal effectiveness of PVP-I, and the efficacy of PVP-I in aquaculture water is still unknown. In this paper, disinfection assays were conducted to evaluate the effectiveness of PVP-I against the A. hydrophila in different types of water. We found that the effective germicidal concentration of PVP-I in outdoor aquaculture water was 25 ppm for 12 h. In indoor aquaculture water with 105 CFU/mL bacteria, 10 ppm and 20 ppm of PVP-I could kill 99% and 100% of the bacteria, respectively. The minimal germicidal concentration of PVP-I in Luria-Bertani broth was 4,000 ppm. Available iodine content assay in LB solutions confirmed that the organic substance had negative impact on the effectiveness of PVP-I, which was consistent with the different efficacy of PVP-I in different water samples. Acute toxicity tests showed that the 24 h-LC50 of PVP-I to swamp eel was 173.82 ppm, which was much higher than the germicidal concentrations in outdoor and indoor aquaculture water, indicating its safety and effectivity to control the A. hydrophila. The results indicated PVP-I can be helpful for preventing the transmission of A. hydrophila in swamp eel aquaculture.