A post-COVID-19 rebound of macrolide-resistant Mycoplasma pneumoniae infections was observed in a teaching hospital in Shenzhen, China. After the relaxation of pandemic control measures, SARS-CoV-2 rebounded rapidly, followed by a slower rebound of M. pneumoniae, peaking 5 months later. The hospitalization rate for M. pneumoniae was lower than that for respiratory viruses, but the 4094 cases of M. pneumoniae within 6 months posed a significant burden on healthcare services. Multivariate analysis showed that adults with M. pneumoniae had more fatigue, comorbidities, and higher CRP levels, while children had higher rates of co-infection with other pathogens and were more likely to be female. Over 85% of M. pneumoniae-positive specimens had mutations at the 23S rRNA gene, with 99.7% showing the A2063G mutation. Patients not treated with effective antibiotics had longer defervescence times. The study highlights the need for updated treatment protocols for M. pneumoniae, especially in children, due to high macrolide resistance rates. The delayed rebound of M. pneumoniae was attributed to factors such as its slow growth rate, long incubation period, and the absence of community circulation during the pandemic. The study also found gender differences in disease susceptibility, with more male cases in children and more female cases in adults. The findings emphasize the importance of targeted next-generation sequencing for detecting macrolide-resistant strains and the need for appropriate antibiotic treatment. The study underscores the significance of monitoring and managing M. pneumoniae infections in the post-pandemic era.A post-COVID-19 rebound of macrolide-resistant Mycoplasma pneumoniae infections was observed in a teaching hospital in Shenzhen, China. After the relaxation of pandemic control measures, SARS-CoV-2 rebounded rapidly, followed by a slower rebound of M. pneumoniae, peaking 5 months later. The hospitalization rate for M. pneumoniae was lower than that for respiratory viruses, but the 4094 cases of M. pneumoniae within 6 months posed a significant burden on healthcare services. Multivariate analysis showed that adults with M. pneumoniae had more fatigue, comorbidities, and higher CRP levels, while children had higher rates of co-infection with other pathogens and were more likely to be female. Over 85% of M. pneumoniae-positive specimens had mutations at the 23S rRNA gene, with 99.7% showing the A2063G mutation. Patients not treated with effective antibiotics had longer defervescence times. The study highlights the need for updated treatment protocols for M. pneumoniae, especially in children, due to high macrolide resistance rates. The delayed rebound of M. pneumoniae was attributed to factors such as its slow growth rate, long incubation period, and the absence of community circulation during the pandemic. The study also found gender differences in disease susceptibility, with more male cases in children and more female cases in adults. The findings emphasize the importance of targeted next-generation sequencing for detecting macrolide-resistant strains and the need for appropriate antibiotic treatment. The study underscores the significance of monitoring and managing M. pneumoniae infections in the post-pandemic era.