Background The elimination of virulence factor is a difficult problem in the treatment of Pseudomonas aeruginosa (PA) infection. Low temperature plasma (LTP) as a physical therapy technique, which commonly used for inactivation of microorganisms. However, the effects on virulence factors have not been reported.Objective To observe the effect of LTP on PA virulence factors in vitro and explore the related mechanisms, and compare the clinical efficacy with medical ultraviolet (UV). Methods The thawed PA was prepared with a certain concentration of bacterial suspension and constructed biofilm models in vitro, which were randomly divided into LTP and UV treatment group. Meanwhile, The LTP group was subgrouped according to the irradiation gradient of 0 min/5 min/10 min/15 min/20 min. The model of PA biofilm was counted using plate counting method. The biofilm and elastase were quantified using crystal violet and Congo red staining. The levels of reactive oxygen species (ROS) were detected using H2DCFDA fluorescent staining and pH was recorded by pH meter.Results Plate counting showed that: the biofilm PA decreased with the increase of low temperature plasma exposure time (P < 0.001). Compared with UV, the biofilm PA treated with LTP for 5 min was lower (P < 0.001). Crystal violet staining and Congo red staining showed that: the biofilm formation and elastase secretion decreased with the increase of low temperature plasma exposure time, and there was a statistical difference among groups (P < 0.001). Compared with UV, the formation of PA biofilm treated with LTP for 10 min was lower (P=0.005), and the secretion of elastase treated with LTP for 5 min was lower (P=0.049). Microenvironment characteristics showed that: the intracellular ROS increased and the solution pH decreased with the increase of low temperature plasma exposure time, and there was a statistical difference among groups (P < 0.001). Compared with UV, intracellular ROS was higher (P=0.002) and pH was lower after LTP exposure 5 min (P=0.034). Conclusion LTP can inhibit the formation of PA biofilm and elastase by changing the microenvironment which PA is located.