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To evaluate the possible in vitro interaction between natural extracts of watercress (Nasturtium officinale R. Br.) and 2-phenylethyl isothiocyanate, a natural compound derived gluconasturtiin largely present in watercress tissues, with a standard antibiotic, a synergy study was carried out against 11 isolates of extended-spectrum β-lactamases-Escherichia coli. Aqueous and methanolic watercress extracts and 2-phenylethyl isothiocyanate were combined with the antibiotic, and a disc diffusion assay and minimum inhibitory concentration methods were used to assess the in vitro antibacterial activity. The results of this study showed that there is an increase in antibacterial activity of the antibiotic when it was combined with plants extracts and pure compounds. The most interesting result was the combination between 2-phenylethyl isothiocyanate and the antibiotic. Synergistic effects of the antibiotic with watercress extracts and 2-phenylethyl isothiocyanate suggest the potential of these plants and their natural compounds to improve the performance of the antibiotics and could be an interesting tool for antimicrobial therapy. The results led us to conclude that watercress has important pharmacological substances which can be used for developing new and effective antimicrobial agents.Significance and Impact of the Study: This in vitro study intends to demonstrate the potential use of watercress extracts and 2-phenylethyl isothiocyanate as antimicrobial tools against extended-spectrum β-lactamases-Escherichia coli and to determine their ability to act synergistically with commercial standard antibiotics. We intended to increase the knowledge about different clinical and pharmaceutical approaches to fight against E. coli rather than the traditional use of antibiotics. The results may be useful to those involved in the pharmaceutical, biochemical and microbiology industry and/or academic research in terms of developing alternative control measures and efficient intervention methods.