Objective: This in vitro study aimed to evaluate and compare the antimicrobial efficacy of Ampicillin with Ceftriaxone(AC),Diclofenacsodium(DS),TripleAntibioticPaste(TAP),andCal ciumHydroxide(CH)against Enterococcus faecalis in root canal preparations. MaterialsandMethods:Minimuminhibitoryconcentrationsofthemedicaments wereassessedusingagarwell diffusiontechnique.TheMICvaluesforAC,DS,TAP,andCHweredetermined. Theantimicrobialeffectofthe synergy of Ampicillin with Ceftriaxone was evaluated, focusing on its inhibitory concentration against E. faecalis.Antimicrobial activity of AC, DS, TAP, and CH was compared based on colony-forming unit (CFU) counts before and after intracanal medicament application.The difference in bacterial viability was observed using confocal laser microscopy on root canal sections treated with the respective medicaments. Results:MIC results indicated lower concentrations for AC and TAP, suggesting higher efficacy. AC demonstrated the most significant reduction in CFU counts compared to DS, TAP, and CH. Confocal laser microscopy further supported the superior antimicrobial activity of AC. Conclusion(s):The combination of Ampicillin with Ceftriaxone (AC) exhibited the most potent antimicrobial activity against Enterococcus faecalis, even at lower concentrations, when compared to Diclofenac sodium, Triple Antibiotic Paste,andCalcium Hydroxide.Thesefindings suggest thepotential effectiveness of ACas an intracanalmedicamentinendodonticapplications,emphasizingtheneedforfurt herinvestigationsonitsduration of action and effects on other bacteria.