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Background: The distribution of bacteria isolated from bloodstream infections and cumulative antimicrobial susceptibility data are the basis for empirical decisions regarding antibiotics as an initial treatment. Therefore, it is important to consistently collect blood culture results of individual patients and analyze them correctly. Methods: The blood culture results of patients at Gachon University Gil Medical Center from 2016 to 2020 were analyzed retrospectively to determine the bacterial distributions and antibiotic resistance patterns. Duplicates were eliminated by including only the first isolate of each species per patient. Results: Escherichia coli (27.1%) was the most commonly isolated bacterium from blood cultures, followed by Klebsiella pneumoniae (10.1%) and Staphylococcus aureus (8.6%). The methicillin resistance rate of S. aureus was 49.2%, and the vancomycin resistance rate of Enterococcus faecium was 39.5%; with no significant changes over the study period. The cefotaxime, ciprofloxacin, and ertapenem resistance rates of E. coli were 35.0%, 46.8%, and 0.7%, respectively. Seventeen carbapenem-resistant E. coli strains were isolated, of which 11 produced carbapenemase. The cefotaxime, ciprofloxacin, and ertapenem resistance rates of K. pneumoniae were 29.5%, 31.7%, and 5.4%, respectively. Forty-eight carbapenem-resistant K. pneumoniae strains were isolated, of which 37 produced carbapenemase. The imipenem resistance rates of Acinetobacter baumannii and Pseudomonas aeruginosa were 72.3% and 23.4%, respectively. Conclusion: In the blood culture results from 2016 to 2020, the isolation frequency of E. coli, K. pneumoniae, and E. faecium showed an increasing trend, whereas that of S. aureus was stable. Over the 5 year study period, the ciprofloxacin resistance rate of E. coli and P. aeruginosa and ampicillin/sulbactam resistance rate of A. baumannii significantly increased.