Objective To investigate the positive rates of Mycoplasma pneumoniae (MP) and other pathogens in children with acute respiratory infection (ARTI) before, during and after COVID-19 pandemic in Shanghai. Methods The clinical data of children with ARTI who underwent respiratory 5-plex and influenza 3 plex tests in Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine from January 1, 2019 to December 30, 2023 were retrospectively analyzed. Results A total of 91825 ARTI cases were included, including 48729 male cases and 43096 female cases, and the median age was 5 (3-8) years old. Before the epidemic, there were 14096 cases, with 5126 positive cases of respiratory pathogens (36.4%); during the epidemic, there were 13366 cases, with 2963 positive cases (22.2%); and after the epidemic, there were 64363 cases, with 33510 positive cases (52.1%). There were significant differences in male proportion, age distribution, and positive rates of various pathogens among the pre-epidemic, epidemic, and post-epidemic groups of ARTI patients (P<0.05). The proportion of children>6 years old was higher in the pre-epidemic and post-epidemic groups, while the proportion of 0-3 years old was higher in the epidemic group (P<0.014). The positive rates of MP in various years and months between 2019 and 2023 showed statistical significance (P<0.05). The positive rate of MP was higher in January, February, and December 2019 (20.3%-37.7%), as well as in January to March 2020 (21.5%-35.3%). In 2021, only the positive rate of MP in December (25.8%) exceeded 20.0%, while all rates in 2022 were below this threshold. From April to June and August to December 2023, the positive rate ranged from 21.5% to 37.2%. The number of MP-positive cases was 23247, and there were statistically significant differences in gender and age distribution among the groups before, during, and after the epidemic (P<0.05). The proportion of MP-positive children>6 years old was higher in the pre- and post-epidemic groups, and the proportion of 0-3 years old in the epidemic group was higher (P<0.014). Conclusions During the period from 2019 to 2023, there have been certain alterations in the epidemiological characteristics of various pathogens associated with ARTI, particularly MP. Moreover, significant changes have occurred in both age distribution and seasonal epidemiological patterns of MP.