Eryngium aquaticum L. (Apiaceae), a culinary herb enjoyed in global cuisines, is also valued for its medicinal properties. The essential oil (EO) extracted from E. aquaticum leaves by hydro-distillation was subjected to antibacterial and antibiofilm activity using a microtiter plate-based in vitro assay against Staphylococcus aureus and Pseudomonas aeruginosa. Compounds identified by GC-MS analysis of EO were screened against the transcriptional regulatory proteins SarA of S. aureus and LasR of P. aeruginosa by molecular docking analysis. The minimum inhibitory concentration (MIC) was recorded as 250 μg/mL against both of these two pathogens. The EO of E. aquaticum also showed concentration-dependent antibiofilm activity against these pathogens, with a maximum inhibition of 50.9 and 48.03% against P. aeruginosa and S. aureus, respectively, at the highest concentration (500 μg/mL) tested. The GC-MS analysis identified 17 compounds and all of them showed moderate to weak binding affinity for the active sites of SarA and LasR, with pentanedioic acid (2,4-di-t-butylphenyl) mono-ester showing the best docking score against SarA (-5.7 kcal/mol) and LasR (-8.0 kcal/mol). This study suggests that E. aquaticum can be a good source of EO with antibacterial and antibiofilm activity against P. aeruginosa and S. aureus.