Objectives: Extended-spectrum beta-lactamases (ESBLs) have been detected more frequently in members of the Enterobacteriaceae family, particularly Escherichia coli and Klebsiella pneumoniae. Infections caused by ESBL-producing bacteria are often resistantto treatment with various antibiotic classes and accompanied by increased complication risks, mortality, and costs. In this study,blood culture results were analyzed to determine the change in the ESBL production rate and antibiotic susceptibilities in E. coliand K. pneumoniae isolates over a period of 3 years.Methods: The results of blood cultures sent to our laboratory between February 2014 and August 2016 were examined retrospectively. Repeat isolates from the same patient were not included when antibiotic susceptibility rates and clinical distributions werecalculated. A BD Bactec FX automated blood culture system (Becton Dickinson and Company, Franklin Lakes, NJ, USA) was used toexamine the blood cultures. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (Product name?; BrukerDaltonics, Inc., Billerica, MA, USA) was used to identify microorganisms. For antibiotic susceptibility tests (AST) and ESBL detection,the Kirby Bauer disk diffusion method or a Phoenix automated system (Becton Dickinson and Company, Franklin Lakes, NJ, USA)was used. When the AST results were evaluated, Clinical and Laboratory Standards Institute breakpoints were used for 2014 and2015, and European Committee on Antimicrobial Susceptibility Testing breakpoints were used for 2016.Results: During the 3-year period, 224 (35%) of 632 E. coli and 137 (31%) of 439 K. pneumoniae isolates were determined to be ESBL-producers. The ESBL-positive isolate percentage for E. coli and K. pneumoniae for 2014, 2015, and 2016 was 23%, 36%, 48% and23%, 32%, 37%, respectively. The increase in ESBL was statistically significant for both E. coli (p<0.001) and K. pneumoniae (p=0.011).ESBL-positive E. coli and K. pneumoniae strains were most sensitive to carbapenem-class antibiotics, amikacin, and colistin. Whilethere was no meropenem-resistant strain, 5 (3.3%) ertapenem-resistant and 1 (0.7%) imipenem-resistant ESBL E. coli strains weredetected. The ESBL K. pneumoniae strain resistance rate to ertapenem, imipenem, and meropenem was 12%, 11.2%, and 11.1%,respectively. The resistance rates of K. pneumonia strains to ertapenem, imipenem, meropenem, and piperacillin-tazobactam increased significantly over the study period (p<0.001).Conclusion: Monitoring ESBL rates and the antibiotic susceptibility of E. coli and K. pneumoniae strains of bloodstream infectionsis of the utmost importance in guiding empiric antibiotic therapies and patient management.