Enterococcusfaecalis(E.faecalis)isamicroorganismthatcansurviveextremechallengesinobturatedrootcanals.Theaimofthisstudywastoevaluatetheefficacyofanon-thermalatmosphericpressureplasmaplumeagainstE.faecalisinvitro.Anon-thermalatmosphericpressureplasmajetdevicewhichcouldgenerateacoldplasmaplumecarryingapeakcurrentof300mAwasused.TheantibacterialefficacyofthisdeviceagainstE.faecalisanditsbiofilmunderdifferentconditionswasdetected.TheantibacterialefficacyoftheplasmaagainstE.faecalisandStaphylococcusaureus(S.aureus)wasalsoevaluated.Afterplasmatreatment,theaveragediameterofinhibitionzoneonS.aureusandE.faecaliswas2.62±0.26cmand1.06±0.30cm,respectively(P<0.05).Thediameterwasincreasedwithprolongationofthetreatmentduration.ThediametersofinhibitionzoneofthesealedPetridisheswerelargerthanthoseoftheuncoveredPetridishes.Therewassignificantdifferenceincolony-formingunitsbetweenplasmagroupandcontrolgrouponE.faecalisbiofilm(P<0.01).ThetransmissionelectronmicroscopyrevealedthattheultrastructuralchangescytodermofE.faecaliswereobservedaftertreatmentfor2min.Itisconcludedthatthenon-thermalatmosphericpressureplasmacouldserveasaneffectiveadjuncttostandardendodonticmicrobialtreatment.