Research conducted in this study shows the applied in vitro antimicrobial and antibiofilm activity of the four extracts isolated from Trapa natans L. leaves. In this study, different methods were used (microdilution, tissue culture plate, different colorimetric methods, GC-FID and GC-MS analysis). While the water extract didn't show antibacterial activity, the acetone extract showed the strongest one. The same activity in the case of Pseudomonas aeruginosa (MIC was 313 μg/mL) was better than the activity of controls and it matched with antibiofilm activity. The effect of extracts was better on G+ bacteria (MICs were <78-625 μg/mL). For ethanol and ethyl acetate extracts all BIC values were better than MICs. Extracts showed a significant effect on Aspergillus restrictus (MICs were < 78/156 μg/mL). The GC and GC-MS analysis of the ethyl acetate extract revealed the identification of 22 compounds with (all E)-squalene (20.2%), n-alkanes and norlignan hinokiresinol among the most abundant ones. This is the first time that T. natans was studied using these methods.