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We determined an antibiotic resistance mechanism in the eastern region, KSA, and the genetic factor clonal relatedness within Gram-negative bacteria. During our retrospective study, a total number of 29 <i>E. coli</i> ESBL producer strains were isolated for patients visiting King Fahad Hospital, Al-Ahsa, KSA. The <i>bla</i> genes were detected via PCR and identified via sequencing. Associated plasmid-mediated quinolone resistance genes, as well as <i>int1</i> and <i>int2</i> genes, were also studied. Phylogenetic groups, the ST131 clone, virulence factors, and PFGE were also checked. The <i>bla_CTX-M-9</i> (3.7%), <i>bla_CTX-M-27</i> (22.2%), and <i>bla_CTX-M-15</i> (77.8%) genes were identified; however, the <i>bla_CMY-42</i> (7.4%) gene was recorded for the first time in KSA. The <i>qnrS1</i> gene was found in 44.4% of strains, and among them, 50% concomitantly harbored the <i>aac(6′)Ib-cr</i>. The <i>int1</i> gene was detected in 25.9% strains; nonetheless, the <i>int2</i> gene was found in 7.4% of isolates. The strains belonged mainly to the B2 and D phylogroups. PFGE showed unrelated patterns. Some isolates belonged to the pandemic clone ST131. We describe a large dissemination of antibiotic resistance to third-generation cephalosporins in the eastern region, KSA, with the occurrence of the <i>bla_CMY-42</i> gene. The clone ST131 seems to be the principal contributor for <i>bla_CTX-M-15</i> gene spread.