Catheter-related bloodstream infections (CRBSIs) are associated with significantly increased morbidity and mortality. They are usually caused by microorganisms that embed themselves in a biofilm layer on the catheter surface. The present study aimed at comparing the in-vitro activity of Antibiotic Lock Technique (ALT) using ciprofloxacin, gentamicin, EDTA and ethanol to determine the optimal concentration and adequate duration of locking therapy for treatment of staphylococcal biofilm model developed on a central venous catheter model. The biofilm producing ability of twenty- nine Staphylococci isolates collected from different labs was tested using the Tissue Culture Plate method (TCP) method to choose nine biofilm producing isolates (3 MRSA, 3 MSSA, and 3 MRCoNS) to undergo the ALT experiment.Among the agents used, only gentamicin 5 mg/ml was able to eradicate all types of staphylococcal biofilms after 72hrs. Gentamicin 1mg/ml was able to eradicate only MRSA biofilms after 72 hrs. Ethanol 20% was able to eradicate only MRCoNS biofilms after 24hrs. However, ciprofloxacin and disodium EDTA couldn't eradicate any of the biofilms developed in this study except for the ciprofloxacin 2 mg/ml that could eradicate MRCoNS biofilm after 72 hrs.Further clinical studies are required to verify the results of our in- vitro experiment.