Resistance to already known antibiotics is a burning issue. Today, plant material is increasingly being tested with the aim of detecting pharmacologically active components. Watermelon seeds are most often discarded as waste, and bearing in mind that other parts of the watermelon contain a lot of bioactive components, the logical sequence is also the examination of the seed extract. The aim of this paper is to examine the content of bioactive components, antibiotic activity, minimum inhibitory concentration (MIC) as well as antibiofilm activity extracts of seeds of two species of watermelon, from Croatia and Greece, obtained from two different extraction methods, maceration and Soxhlet extraction. Phytochemical analysis was performed using three different HPLC methods. The antibacterial effect was done using disc method, MIC was determined using broth dilution method and biofilm formation was done using Tryptic Soy Broth (TSB). The results indicate the presence of some flavonoids in all samples, as well as on antimicrobial activity against certain strains of bacteria. The best result iwas shown by the ethanol extract from Greece and overall ethanol extracts show slightly better antimicrobial activity compared to methanol extracts. Depending on the tested concetration, different biofilm categories appear. The results show that as the extract concentration decreases, the biofilm formation capacity increases. Our data show that extracts of the seeds have the potential to act as antimicrobial agents, and further research is needed.

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