Objective: To investigate the distribution of bacteria and analyze its drug resistance change from the midstream urine in the First Affiliated Hospital of Nanjing Medical University in 2014, so as to provide experimental basis for clinical rational use of antibiotics. Methods: Automatic detection machine of VITEK 2 Compact or API system was used for identification of bacteria and fungus. Kirby-Bauer method was used to test bacteria drug sensitivity and ATB FUNGUS 3 was used to test fungal drug sensitivity. Whonet 5.6 software was used for statistical analysis. Results: In total, 5 172 samples of midstream urine were collected during the year of 2014, and 1 326 ones of them were positive for culturing of pathogenic microorganisms (positive rate, 25.64%). The proportions of pathogenic bacteria were 72.0% for gram negative bacilli, 19.6% for gram positive coccus and 8.4% for fungus respectively. Escherichia coli remained to be one of the most commonly found pathogenic bacteria, of which the proportion of Escherichia coli that producing ESBLs was 61.3%. High sensitivities were also observed with respect to carbapenems for gram negative bacilli, vancomycin and linezolid for gram positive coccus, and amphotericin B and voriconazole for fungus. Besides, three strains of and five strains of Klebsiella pneumoniae resistant to imipenem and meropenem; five strains of Enterococcus faecium resistant to vancomycin were also detected. Conclusion: Escherichia coli is one of the major pathogenic bacteria that result in the urinary system infection. Various kinds of pathogenic microorganisms are becoming increasingly resistant to antibiotics that commonly used clinically.