Ureaplasma urealyticum (U. urealyticum) is a very important pathogenic microorganism. In view of a worsen resistance of U. urealyticum to antibiotics recently, it’s in great need to exploit new alternative therapies for U. urealyticum infections. The aim of this research is to study the in vitro susceptibility of U. urealyticum to toluidine blue-mediated photodynamic antimicrobial chemotherapy (PACT). Two biovars of U. urealyticum including two standard strains and two clinical strains were incubated with photosensitizer toluidine blue with different concentrations from 2.5 to 0.039 062 5 mmol/L. After 20 or 60 min incubation, they were treated with (633±10) nm red light source, with four different light fluences including 48, 102, 204 and 408 mJ/cm². The association between the incubation time, toluidine blue concentration, as well as the light fluence and PACT effect on U. urealyticum was evaluated 48 h after treatment. Besides, the difference in response to PACT between the two biovars of U. urealyticum was also analyzed. The results showed that PACT had an inactivation effect on the growth of U. urealyticum, in a toluidine blue concentration-dependent and fluence-dependent manner. Longer incubation time induced more extensive inactivation of U. urealyticum. No significant difference in susceptibility to PACT was observed between the two biovars of U. urealyticum. It is concluded that toluidine blue-mediated PACT can inactivate U. urealyticum in vitro, and this therapy may become a promising alternative treatment for U. urealyticum infections.