Urinary tract infections are common diseases and Escherichia coli is the most common cause of this disease, especially in infants and children. The 200 samples were collected for the period from July to December 2020 from the Central Children’s Hospital in Baghdad from infants and children and cultured on MacConkey and blood agar for primary isolation of E. coli bacteria. The VITEK 2 system was used for biochemical tests and final identification. Antibiotic susceptibility for 16 antibiotics was determined using the VITEK 2 system, as well as the ability for biofilm formation using the tissue culture plate method, while DNA was isolated for molecular antibiotic resistance genes tetA, tetB, CITM, sul1, sul2, and sul3 using the standard polymerase chain reaction. Results showed that 40/62 isolates (64%) belonged to E. coli bacteria (32 females and 8 males), and isolates were of varying resistance to antibiotics, with the highest resistance to ampicillin and sulfamethoxazole and the lowest resistance to tigecycline and most MDR. The percentage of biofilm formation ability was 62.5%. The ratio of resistance genes CITM, tetA, tetB, sul1, and sul2 for E. coli bacteria was 5%, 45%, 12.5%, 35%, and 40%, respectively, while the sul3 gene did not appear in isolation from any clinical local isolation. So, E. coli isolates isolated from children showed a high extent of antibiotic resistance as the genetic sequencing of resistance genes showed that there were many mutations with a type of transmission or silence or transition. In conclusion, E. coli isolated from children was highly resistant to antibiotics and had the ability to form biofilms as well as the presence of a resistance gene that could be transmitted among bacteria and spread antibiotic resistance.