Introduction This study explores the intricate relationship between bacterial flora and the occurrence of Escherichia coli (E. coli) infections in gynecological patients. It aims to provide insights into the various treatment strategies used to effectively manage bacterial pathogens, especially E. coli infections. By conducting a comprehensive analysis of the bacterial flora in gynecological patients, the study highlights the notable presence of E. coli, promptingfurther investigation into the factors that contribute to its colonization. The objective of the study is to comprehensively investigate and detect urinary tract infections (UTIs) specifically caused by E. coli among gynecological patients. The study aims to delve into bacterial flora prevalence, antibiotic resistance patterns, and potential virulence factors. Through this analysis, the study intends to identify effective strategies for rapid detection and diagnosis of UTIs caused by E. coliby utilizing advanced microbiological and molecular techniques. Furthermore, the study aims to formulate and propose a strategic treatment approach with a particular emphasis on selecting appropriate antibiotics to reduce the risk of severe infections and associated complications. Materials and methods The methodology employed in this study included the isolation and characterization of bacterial strains from clinical samples obtained from gynecological patients. A total of 52 urine specimens were collected from patients with complaints of infection in the urinary tract and infertility. These samples underwent both preliminary and confirmatory microbiological analysis, such as gram staining, biochemical confirmation test, and antibiotic susceptibility, and further proceeded with the multiplex polymerase chain reaction (PCR) technique. The results of PCR and antibiotic susceptibility revealed the specific gene involvement and resistant characteristics of E. coli. Results The findings revealed a total of 32 specimens positive for E. coli, of which 10 patients had infertility complaints and 22 patients had UTIs. The preliminary test, gram staining, showed the gram-negative bacilli E. coli, and the nutrient agar plate revealed smooth circular translucent colonies; MacConkey agar showed pink-colored lactose-fermented colonies; and the blood and chocolate agar plates showed grayish white moist gamma-hemolytic colonies. The biochemical confirmation of E. coli resulted in positive for indole and methyl red tests and negative for Voges-Proskauer and citrate utilization tests. The multiplex PCR analysis confirmed the E. coli strains with the presence of two target genes,stx2d and stx2e. Conclusion To summarize, this study offers valuable insights into the bacterial flora of gynecological patients impacted by E. coli infections, which provides a foundation for the development of precise and efficient treatment strategies. The results emphasize the importance of personalized treatment approaches that consider both the microbiological characteristics of the infection and the evolving landscape of antibiotic resistance. The implication of this research extends to enhancing clinical outcomes and alleviating the burden of E. coli infections in gynecological settings.