Objective To understand the isolated strains and antimicrobial susceptibility testing results of strains from hospitalized patients before the first therapeutic use of antimicrobial agents, provide reliable microbiological basis for rational use of antimicrobial agents. Methods Based on the data in antimicrobial clinical decision support system as well as isolated strains and antimicrobial susceptibility testing results in laboratory information system in a hospital, clinically isolated strains before the first therapeutic use of antimicrobial agents after patients' admission and strains isolated from all clinical specimens during patients' hospitalization period from 2015 to 2018 were collected, distribution and drug resistance of bacterial strains before and after antimicrobial use were compared. Results From 2015 to 2018, 69 037 non-repetitive strains (including bacteria and fungi) were isolated from all specimens of hospitalized patients, including 17 900 (25.93%) strains of Gram-positive bacteria, 44 055 strains (63.81%) of Gram-negative bacteria and 7 082 strains (10.26%) of fungi. 15 017 non-repetitive strains were isolated from specimens collected before the first therapeutic use of antimicrobial agents after patients' admission, 4 661 strains (31.04%) of which were Gram-positive bacteria, 9 451 (62.93%) were Gram-negative bacteria and 905 (6.03%) were fungi. The top 5 isolated strains from specimens before therapeutic antimicrobial use were Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa and coagulase negative Staphylococcus, the top 5 isolated strains from all specimens were Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Candida spp. and Escherichia coli; the proportion of fastidious bacteria such as Streptococcus spp., Haemophilus influenzae and Moraxella catarrhalis was lower among all detected specimens. The common clinical isolates included Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii. Drug resistance rates of strains from specimens after antimicrobial therapy were higher. Conclusion This study is the first report of clinically isolated strains from large sample before the first therapeutic use of antimicrobial agents, the common clinical isolates include Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii, specimens shows higher drug resistance after antimicrobial therapy.