The immediate killing effect of atmospheric-pressure plasma (APP) has been frequently investigated, but its sustained killing activity is poorly understood. The goal of the present study is to evaluate both the immediate and sustained killing effects of APP on Enterococcus faecalis. The APP jet was evaluated by optical emission spectroscopy (OES) and laser-induced fluorescence spectroscopy (LIF). Hydroxyapatite (HA) discs coated with bovine dermal type I collagen were used as substrates for bacterial growth. After the formation of E. faecalis biofilms on the HA discs for seven days or three weeks, the samples were treated with (A) 2 ml of saline, (B) APP, or (C) 2 ml of 2% chlorhexidine digluconate (CHX) for 5 min. The treated samples were then cultured for three or seven days, after which they were examined by scanning electron microscopy and confocal laser scanning microscopy. The OES results showed that typical reactive oxygen and nitrogen species were included in the full spectrum. The fitted curve indicated that the rotation temperature of N-2 was close to room temperature. The LIF results showed that the maximal O and OH intensities occurred at 5 mm from the nozzle. For both the seven-day and three-week biofilms, the CHX and APP treatments had significant sterilization results (P < 0.05) compared to the saline group in terms of immediate and sustained killing effects. APP demonstrated excellent potential for use as an alternative approach for the treatment of periapical diseases.