Shiga toxin-producing Escherichia coli (STEC) is a major foodborne pathogen associated with dairy products,
while the increasing prevalence of multidrug-resistant (MDR) strains has intensified the need for effective natural
antimicrobial alternatives. This study investigated the occurrence, molecular characteristics, antimicrobial
resistance, and virulence profiles of STEC isolated from raw milk, yogurt, and Kareish cheese, and evaluated the
antibacterial efficacy of a thyme essential oil nanoemulsion. Nineteen E. coli isolates were recovered, with
isolation rates of 26.66%, 23.33%, and 13.33% from raw milk, yogurt, and Kareish cheese, respectively. Antimicrobial
susceptibility testing revealed high resistance to ampicillin (100%), sulfamethoxazole-trimethoprim
(94.7%), penicillin (89.5%), and amoxicillin (78.9%), with five isolates exhibiting multidrug-resistant phenotypes.
Molecular analysis identified the stx1 gene in 42.1%, 15.78%, and 21.05% of isolates from raw milk,
yogurt, and Kareish cheese, respectively, whereas eaeA was detected in only 10.5% of isolates and stx2 was not
detected. GC–MS analysis identified thymol (37.5 ± 1.2%) and carvacrol (15.0 ± 0.8%) as the predominant
bioactive constituents of thyme essential oil. The nanoemulsion exhibited a mean hydrodynamic diameter of
135.7 ± 23.04 nm, a PDI of 0.251, spherical morphology with a core particle size of 35.45 nm, and significantly
enhanced antibacterial activity compared with the free essential oil by reducing the minimum inhibitory concentration
from 0.19 ± 0.006 to 0.009 ± 0.0006 mg/mL (P < 0.001) and increasing the inhibition zone from
29 ± 1 to 32 ± 1 mm. Scanning electron microscopy confirmed extensive membrane disruption and severe
morphological damage in treated bacterial cells. Data were analyzed using one-way analysis of variance
(ANOVA) followed by Tukey's multiple comparison test. Collectively, these findings demonstrate that thyme
essential oil nanoemulsion is a promising clean-label antimicrobial strategy for controlling multidrug-resistant
STEC and improving the microbiological safety of dairy products.