| Summary: | This study aimed to investigate the prevalence and antimicrobial resistance of enterococci- and ESBL-producing <i>E. coli</i> isolated from milk of bovine mastitis cases in Egypt. Fifty milk samples of dairy animals were collected from localities in the Nile Delta region of Egypt. Isolates were identified using MALDI-TOF MS, and antibiotic susceptibility testing was performed by the broth microdilution method. PCR amplifications were carried out, targeting resistance-associated genes. Seventeen <i>Enterococcus</i> isolates and eight coliform isolates could be cultivated. Vancomycin resistance rate was high in <i>Ent. faecalis.</i> The VITEK 2 system confirmed all <i>E. coli</i> isolates as ESBL-producing. All <i>Ent</i>. <i>faecalis</i> isolates harbored <i>erm</i>(B), <i>tet</i>L and <i>aac-aph</i>D genes. The <i>van</i>A gene was detected in <i>Ent. faecalis</i> isolate, <i>van</i>B was found in other <i>Enterococcus,</i> while one isolate of <i>E. casseliflavus</i> exhibited the <i>van</i>A gene. <i>E. coli</i> isolates exhibited high prevalence of erm(B) and tetL. <i>E. coli</i> isolates were analyzed by DNA microarray analysis. Four isolates were determined by O-serotyping as O8 (n = 1), O86 (n = 2) and O157 (n = 1). H-serotyping resulted in H11, H12, H21 (two isolates each) and one was of H16 type. Different virulence-associated genes were detected in <i>E. coli</i> isolates including <i>lpf</i>A, <i>ast</i>A, <i>cel</i>B, <i>cma</i><i>hem</i>L, <i>int</i>I1 and <i>int</i>I2, and the <i>iro</i>N gene was identified by DNA microarray analysis.
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