Abstract Sea anemones produce many biologically active compounds including neurotoxins, pore-forming toxins, phospholipases and proteinase inhibitors. The Persian Gulf is an unexplored environment and maybe a rich source of marine natural products. The aim of this study is screening and identification of bioactive metabolites from Stichodactyla haddoni (Haddon’s sea anemone) collected at the Persian Gulf. The crude extract of the sea anemone (tentacle, disc and total body) was obtained by methanol solvent. The antibacterial assays were carried out by the disc diffusion method. The antibiofilm activity (biofilm formation, biofilm destruction and reduction of metabolic activity) of the sea anemone extracts was evaluated by microtiter plate method. The bioactive compounds were identified by GC–MS analysis. Data showed that the best antibacterial effect (relate to P. aeruginosa) is obtained from extracts of “total body” section. Values of minimum inhibitory concentration and minimum bactericidal concentration show that the maximum antibacterial activity takes place at 10–20 mg/ml concentration. Three parts of sea anemone exhibit different inhibition against biofilm of bacteria, in particular, inhibition of biofilm observed by the tentacle, disc and total body against P. aeruginosa, K. pneumonia and A. baumannii, respectively. Biofilm of P. aeruginosa was the most sensitive and the biofilm of B. cereus was the most resistant structure between all pathogenic bacteria. The best reduction in the metabolic activity was observed in P. aeruginosa and K. pneumonia among tested bacteria. Aliphatic compounds were predominant bioactive metabolites in this sea anemone. The marine animal and especially sea anemone produce useful bioactive compounds that can be used to prevent bacterial biofilm; application of bioactive materials, reported in this study, can be proposed for future studies.