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Objective The clinical distribution and drug resistance of Escherichia coli and Klebsiella pneumoniae in a hospital of Nanjing from 2020 to 2022 were analyzed in order to provide the basis for clinical rational use of antibiotics. Methods The distribution and drug resistance of Escherichia coli and Klebsiella pneumoniae specimens in Zhongda Hospital Southeast University from 2020 to 2022 were retrospectively analyzed. Results A total of 3 507, 4 323 and 4 806 pathogenic bacteria were detected from 2020 to 2022. There were 616, 748 and 840 strains of Escherichia coli , and 456, 631 and 757 strains of Klebsiella pneumoniae , with the total 3-year detection rate of 17.4% and 14.6%. There was a statistically significant difference in the constituent ratio of pathogenic bacteria between 2020 and 2022 (P＜0.01). From 2020 to 2022, the top three samples of pathogenic bacteria were sputum, blood and urine. From 2020 to 2022, the drug resistance rates of Escherichia coli to cefepime, ceftriaxone, cefazolin, gentamicin, ciprofloxacin, levofloxacin and compound trimethoprim were significantly different (P＜0.01). The drug resistance rates of Klebsiella pneumoniae to imipenem, ertapenem, cefepime, ceftriaxone, cefoperazone sulbactam, cefazolin, cefoxitin, piperacillin tazobactam, ampicillin, amikacin, gentamicin, levofloxacin, nitrofurantoin and compound trimethoprim were significantly different (P＜0.01). Conclusion The average detection rate of Escherichia coli and Klebsiella pneumoniae was more than 30% in 2020—2022. The resistance of Escherichia coli and Klebsiella pneumoniae to various antibacterial drugs is statistically different. The clinically targeted drugs need to be selected in strict accordance with the drug sensitivity results.