Urinary tract infections (UTIs), caused by Escherichia coli 80% to 85% of the time, are one of the most important causes of morbidity and health care spending affecting persons of all ages. These infections lead to many difficult problems, especially increasing resistance to antibiotic drugs. Bacterial biofilms play an important role in UTIs, responsible for persistent infections leading to recurrences and relapses. In this study, we have investigated the antibacterial activity of five medicinal plant essential oils against UTIs caused by E. coli using disc diffusion and minimal inhibition concentration (MIC) methods. In addition, biofilm inhibitory action of oils was realized by crystal violet. Gas chromatography–mass spectrometry (GC–MS) analysis showed a variability between oils in terms of compound numbers as well as their percentages. Antibacterial activity was observed only in cases of Origanum majorana, Thymus zygis and Rosmarinus officinalis, while Juniperus communis and Zingiber officinale did not showed any effect towards E. coli isolates. T. zygis essential oil demonstrated the highest antibacterial activity against E. coli isolates, followed by O. majorana and R. officinalis. Further, oils showed high biofilm inhibitory action with a percentage of inhibition that ranged from 14.94% to 94.75%. R. officinalis oil had the highest antibiofilm activity followed by T. zygis and O. majorana. Accordingly, tested oils showed very effective antibacterial and antibiofilm activities against E. coli UTIs and can be considered as good alternative for antibiotics substitution.