Objective To explore the clinical value of microbiological examination and bacterial resistance surveillance. Methods A total of 1000 specimens collected from June 2021 to June 2022 in our hospital were used as the research subjects. Clinical microbial test and bacterial resistance monitoring were performed, and the positive rate of specimens and the distribution of pathogenic bacteria were analyzed. There were 232 cases (23.20%) with positive results. The bacterial distribution was as follows: 283 Escherichia coli, 192 Klebsiella pneumoniae, 161 Pseudomonas aeruginosa, 127 Staphylococcus aureus, 90 Acinetobacter baumannii, 76 coagulase-negative staphylococci, 48 Enterobacter spp. and 23 Enterococcus spp. In this study, cefazolin, penicillin, ampicillin, ciprofloxacin, ofloxacin and ceftazidime were selected for research, and penicillin showed the strongest resistance. Klebsiella, Pseudomonas aeruginosa, Enterobacter, Proteus and Acinetobacter were Gram-negative bacteria, and the resistance rate to common antibiotics was less than 70%. The resistance rate of Enterococcus to the above antibiotics is less than 60%. Staphylococci have high drug resistance to ampicillin and penicillin. Conclusion Bacterial resistance monitoring in microbiological examination can master the changes of bacterial resistance, guide the use of antibacterial drugs, and effectively improve the treatment effect.