Acinetobacter baumannii is one of the most prevalent pathogens in nosocomial infections and has been cause of concern in recent years because, it has presented multiresistance to antibiotics and besides can form biofilms on biotic and abiotic surfaces like tissues and medical devices. Therefore, the search for new alternatives of natural origin to inhibit biofilm formation is being conducted. In the present investigation, sub-lethal concentrations (5.61 mg/mL, 3.74 mg/mL and 1.87 mg/mL) of methanolic extract of Nothoscordum bivalve were evaluated, showing biofilm formation inhibition up to 40.8%, in one nosocomial isolated of A. baumannii by the microtiter biofilm formation assay using crystal violet. On the other hand, the concentrations of 5.61 mg/mL and 3.74 mg/mL, caused an overexpression (up to 15.4 times) in the genes involved in the formation of biofilm (abaI, bap and csuE); due to this, the interaction of the extract with the bacteria was analyzed by scanning electron microscopy (SEM) and cellular damage was observed in the structure and stability of biofilm.