Objective To explore the effect of three surfactants (dodecyl hydroxypropyl sulfobetaine, sodium dodecylbenzene sulfonate and cetyl pyridine bromide) on the permeability of Staphylococcus epidermidis (Se) biofilm and the expression of the key biofilm formation gene agr, and provide reference for the clinical use of surfactants to control Se biofilm relevant infection. Methods Se biofilm permeability model was constructed to detect the effect of surfactant treatment on biofilm permeability. The effect of three surfactants on agr transcription in biofilm-forming Se in the suspension state was observed at different time periods, and the possible effect of surfactants on the adhesion and virulence of biofilm-forming Se was assessed at molecular level. Results All three types of surfactants significantly affected the permeability of Se biofilm at minimum inhibitory concentration (MIC) of 2, 1, and 1/2 (P < 0.001), enhancing drug permeability to biofilm. Compared with negative control group, dodecyl hydroxypropyl sulfobetaine had a significant up-regulation effect on agr expression at 0.5, 4 and 12 hours (P < 0.001). Sodium dodecylbenzene sulfonate had a significant down-regulation effect on agr expression at 0.5, 4 and 24 hours (P < 0.001) and a significant up-regulation effect at 12 hours (P < 0.001). Cetyl pyridine bromide had a significant up-regulation effect on agrexpression at 0.5 hours (P < 0.01) and a significant down-regulation effect at 12 and 24 hours (P < 0.001). Conclusion All three surfactants had strong effects on the permeability of Se biofilm, with significant modulating effect on agr expression at different time periods, which may play a role in the formation of Se biofilm, thus preventing the spread of infection and leading to the reduction of bacterial virulence.