Objective To investigate the distribution and antimicrobial resistance characteristics of fastidious bacteria in a children's hospital, and provide reference for pediatricians to use antimicrobial agents rationally. Methods Main species and antimicrobial resistance of fastidious bacteria from respiratory specimens in a hospital from 2018 to 2022 were analyzed through continuous monitoring. Results The top three isolated fastidious bacteria were Streptococcus pneumoniae (Spn), Haemophilus influenzae (Hi), and Moraxella catarrhalis (Mc). The average resis-tance rate of Spn to erythromycin, clindamycin, tetracycline and cefuroxime was high (>75%), while to penicillin, ceftriaxone, and amoxicillin/clavulanic acid was low (< 15%). The average resistance rate of Hi to ampicillin was high (close to 75%), to cefotaxime, cefuroxime, cefaclor and amoxicillin/clavulanic acid was low (< 45%). The average resistance rate of Mc to ampicillin was high (>90%), to the remaining frequently used antimicrobial agents was low (< 30%). The resistance rates of the three types of bacteria to the above mentioned antimicrobial agents over the past 5 years showed statistically significant differences (all P < 0.05). Conclusion Considering the particularity of antimicrobial use in children, and combined with the results of this study, for Spn infections, empirical treatment may be initiated with penicillin, third-generation cephalosporins, or beta-lactamase inhibitors; for Hi infections, empirical treatment may involve second- and third-generation cephalosporins or beta-lactam antibiotics; for Mc infections, empirical treatment can use antimicrobial agents other than ampicillin. Subsequently, the treatment regimen should be adjusted based on the pathogen and drug sensitivity results.