Aeromonas hydrophila is one of the most important aquatic pathogens that cause fish losses and poor product quality in fish farms. The ability of Aeromonas hydrophila to form a biofilm makes this bacterium resistant to antibiotics. Hence, finding new antimicrobials with the potential to inhibit biofilm formation would be valuable. The aim of the present study was to evaluate the antibacterial and antibiofilm activity of the extract from Pseudoalteromonas piscicida, isolated from the sea anemone (Stichodactyla haddoni) against Aeromonas hydrophila. The Microdilution broth method was used to determine the minimum bactericidal concentration (MBC), and minimum inhibitory concentration (MIC) of Pseudoalteromonas piscicida extract. The anti-biofilm ability of this extract against formation of Aeromonas hydrophila biofilm was assessed using crystal violet staining method in 96-well plates. The results showed that the extract of Pseudoalteromonas piscicida inhibited the growth and biofilm formation of Aeromonas hydrophila at MIC=MBIC= 200 mg/ml. In addition, using physical and chemical treatments, it was shown that the active compounds are mainly proteinous in nature and are proteases. The results from the experimental challenge of zebrafish (Danio rerio) with the pathogen demonstrated that oral administration of Pseudoalteromonas piscicida extract in two safe doses of 200 and 400 mg mL^-1 increased survival rates of the fish and decreased colonization of Aeromonas hydrophila in their intestine within 7 days. The results of the present study showed that Pseudoaltromonas picisidia extract contains bioactive compounds with antibacterial and antibiofilm ability against Aeromonas hydrophila and due to its low toxicity in fish, if purified, it can be a good alternative to antibiotics.