The aim of this investigation was to monitor the occurrence of extended-spectrum beta-lactamase (ESBL) E. coli strains along the Upper Litany River Basin (ULRB), genotype ESBL producers, and determine their sensitivity to different essential oils (EOs). Thirty five E. coli strains were isolated and recognized using double-disc synergy test, then studied for the detection of key ESBL genes and Shiga-toxin genes using multiplex polymerase chain reaction (PCR). Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) broth microdilution, beside the time-kill assay were applied to determine the antibacterial activity of the EOs against the ESBL E. coli. EOs activity on biofilms was detected by antibiofilm screening. 22.8% strains were verified as ESBL producers and bla_CTX-M was identified in all strains, then bla_TEM with 50% of prevalence, and there was no detection of bla_OXA, bla_SHV, and both stx₁ and stx₂ genes. Sesame oil revealed the highest antibacterial activity. Followed by Coconut, Lettuce, and Almond oils with a concentration ranging between 6.25% and 50%. Almond, Sesame, and Lettuce oils showed an interesting effect against biofilm formation, and Coconut and Sesame oils were effective against preformed biofilms. Our study showed the antibacterial and antibiofilm effects of EOs against ESBL E. coli.