Background: Cholecystectomy is one of the frequent causes for abdominal surgery. It is generally thought that the antibiotic concentration used should be four to six times than the minimum inhibitory concentration (MIC) to be effective, when it is used as prophylaxis. Cefotaxime is a commonly prescribed agent for surgical prophylaxis. Aim: The aim of this study was to measure the gall bladder tissue and the plasma concentrations of cefotaxime in cholecystectomy and to assess its suitability as a prophylactic agent. Methodology: 24 patients who were undergoing Cholecystectomy were enrolled to collect plasma and gall bladder tissue samples. Cefotaxime levels in the Gall bladder tissue and plasma samples were estimated. These concentrations were compared against the Minimum Inhibitory Concentrations (MICs) of commonly isolated organisms from surgical wound samples. Results and Discussion: Plasma concentrations to MIC ratios were calculated which were in the range of 10 to 21 times higher than MIC. Tissue drug concentrations to MICs ratios were also calculated and were found to be smaller. This indicated that the concentration of the drug in the tissues was less than the MIC during the sampling time. This situation of lesser tissue concentration of the drug might result in drug failure and subsequent infection if contamination occurs during surgery. Conclusion: This study showed that even though cefotaxime remained at a microbicidal concentration in plasma, it was present in the tissues at ineffective concentrations with respect to MIC. These results showed that the practice of using cefotaxime as a prophylactic agent in cholecystectomy may be reconsidered.