Introduction: Peritonitis is a frequently encountered pathology by a general surgeon. The development of antimicrobial agents and the advancement of surgical techniques in the past decade have decreased morbidity and mortality. The complications range from minor wound infection to life-threatening septic shock and Systemic Inflammatory Response Syndrome (SIRS). Usage of appropriate antimicrobial agents that covers gram-positive, gram-negative, and anaerobic organisms provides good recovery in the patients. But inappropriate usage of antibiotics without following guidelines leads to the development of antibiotic resistance and treatment failure. Objectives: To study the microbiological pattern of the peritoneal fluid in culture and sensitivity and to identify the pattern of antibiotic sensitivity for routinely used organisms. Methods: This is cross-sectional study conducted in 50 patients with peritonitis and perforation confirmed on chest x-ray. Peritoneal fluid is isolated and nature of microorganism are characterized along with antibiotic sensitivity. Results: In this study, the most common site of perforation leading to peritonitis is Duodenum followed by the stomach. Mostly secondary to peptic ulcer disease. The most common organism isolated was Klebsiella followed by E.coli, mixed cultures, Proteus, and pseudomonas. Most of the organisms isolated from the peritoneal fluid on culture were sensitive to the Cephalosporin group followed by Fluoroquinolones and macrolides. Conclusion: Appropriate antibiotic use based on the sensitivity pattern is essential to improve the morbidity and mortality in patients who suffered from perforation secondary to peritonitis and to prevent antimicrobial resistance.