The aim of this study was to estimate the prevalence and antimicrobial susceptibility of Ureaplasma urealyticum and Mycoplasma hominis in a comprehensive teaching hospital Shenyang, China over the past 4 years. A total of 1448 individuals with urogenital symptoms underwent mycoplasma testing between April 2016 and March 2020. Detection, identification and antimicrobial susceptibility testing were carried out using Mycoplasma ID/AST kits. The total infection rate of genital mycoplasmas was 37.5% (543/1448 cases) with an observed increase over the past 4 years. The positive rates of all three detected infections, as well as overall infection rate, were significantly higher in females than in males (P < 0.05). A higher positive rate of infection was observed in females aged 25–29 (60.5%), and in the 15–19 years age group (57.7%). The changes observed among all age groups of females were statistically significantly different (P < 0.001). The positive rates of U. urealyticum and M. hominis co-infection among the four seasons during which the survey was carried out were also observed to be statistically different (P = 0.01). More than 70% of U. urealyticum isolates were found to be resistant to ciprofloxacin, and more than 80% of M. hominis isolates were resistant to erythromycin, roxithromycin, azithromycin and clarithromycin. Josamycin, doxycycline and minocycline were most effective against U. urealyticum and M. hominis. Results of this study found increasing rates of U. urealyticum and M. hominis infection over the past 4 years, particularly among younger age groups. U. urealyticum/Mycoplasma hominis screening among younger age cohorts are therefore strongly recommend to preventing the spread of pathogens. Monitoring antimicrobial resistance is important for preventing transmission of resistant strains of infection and for the management of antibiotics.