Background <p>To investigate the epidemiological profile and antimicrobial resistance patterns of genital mycoplasma in Eastern China and provide evidence-based guidance for clinical management.</p> Methods <p>A retrospective analysis was conducted on clinical records, mycoplasma culture results, and antimicrobial susceptibility testing data from patients with suspected urogenital tract infections between 2018 and 2023.</p> Results <p>Among 47,619 suspected infected patients, 20,830 cases tested positive for genital mycoplasma infection, with an overall infection rate of 43.74%. The infection rate of pure <i>Ureaplasma</i> spp. was 37.00%, for pure <i>Mycoplasma hominis</i> (Mh) was 0.66%, and for the co-infections with <i>Ureaplasma</i> spp. and Mh was 6.08%. The infection rate in females (44.00%) was significantly higher than that in males (20.12%), with a statistically significant difference (<i>P</i> &lt; 0.001). The observed changes in each age group showed statistically significant differences (<i>P</i> &lt; 0.001). Seasonally, the infection rate of mycoplasma in spring was slightly higher than that in winter. Regarding drug resistance, genital mycoplasmas generally exhibited a higher resistance rate to fluoroquinolone drugs, while the resistance rates to tetracycline, doxycycline, pristinamycin, and josamycin were relatively low. The average resistance rates to ciprofloxacin and ofloxacin in patients with pure <i>Ureaplasma</i> spp. infections were relatively high, at 83.39% and 66.34%, respectively. And the resistance rates showed an increasing trend year by year (<i>P</i> &lt; 0.001). Patients with pure Mh infections had the highest resistance rate to ofloxacin (80.32%), followed by ciprofloxacin (69.21%), with no significant differences in resistance rates across the years. Patients co-infected with <i>Ureaplasma</i> spp. and Mh had the highest average resistance rates to both ofloxacin and ciprofloxacin, exceeding 90.00%.</p> Conclusion <p>The infection rate of genital mycoplasma in Eastern China is relatively high, predominantly <i>Ureaplasma</i> spp., with significant resistance to fluoroquinolone drugs. It is necessary for the hospital to enhance monitoring for the genital mycoplasma infections and to conduct drug resistance analysis to guide rational medication use and infection control measures.</p> Clinical trial number <p>Not applicable.</p>