Abstract: Objective This study aimed to investigate the biofilm formation patterns of Saprolegnia parasitica ATCC200013 and the effects of the biosurfactant rhamnolipid on the biofilm， to provide a theoretical basis for developing eco-friendly control agents against saprolegniasis.Method The coverslip method was used to from biofilms of Saprolegnia parasitica ATCC200013 on substrates of polyethylene mesh， and a time curve of biofilm formation was plotted. A CCK-8 kit and safranin staining were used to measure the biomass of mycelium and the biofilm biomass and extracellular matrix amount treated with different concentrations of rhamnolipid. The morphological structure of Saprolegnia parasitica ATCC200013 biofilms were observed by scanning electron microscopy （SEM） and confocal laser scanning microscopy （CLSM）. Mature biofilms were treated with various concentrations of rhamnolipid， with the concentration gradients were prepared at various multiples of the minimum inhibitory concentration （MIC） and labelled as 0MIC，1MIC，2MIC，4MIC， and 8MIC， to evaluate their disruptive effects.Result Saprolegnia parasitica ATCC200013 biofilm began to form at 8 h and reached a stable state between 60-72 h. Rhamnolipid treatments significantly inhibited biofilm development： 15.625 mg/L rhamnolipid exhibited 76.44% inhibition rate， while 31.25 mg/L completely prevented biofilm formation. SEM and CLSM observations revealed that as the rhamnolipid concentration increased， the extracellular matrix amount of biofilm was gradually reduced， and mycelium spacing widened. At 15.625 mg/L， both mycelium biomass and extracellular matrix amount were markedly decreased， mycelium morphology was impaired and appeared flattened， and the proportion of impaired and dead cells increased. Rhamnolipid also suppressed the activity of mature biofilms： after the 24 h treatment， the biofilm of 8MIC rhamnolipid completely lost its activity. After 48 h， the biomass of biofilms of 1MIC rhamnolipid was only 12.3%. As rhamnolipid concentration increased， the adhesion rate and hydrophobicity rate of spores on polyethylene meshes decreased gradually. The adhesion and hydrophobicity in the 15.625 mg/L rhamnolipid treatment group were significantly lower than those in the 0 mg/L rhamnolipid treatment group （P<0.05）.Conclusion Rhamnolipid exhibits notable resistance to Saprolegnia parasitica ATCC200013 biofilm. Rhamnolipid could impairs the drug tole-rance of the biofilm through reducing extracellular matrix production， thereby increasing the sensitivity of Saprolegnia parasitica to antifungal agents. Alternatively， by suppressing extracellular matrix production， rhamnolipid may expose the mycelium to unfavorable conditions， ultimately weaken its survival ability and inducing cell death.