Background & Objectives: The familiarity on the part of the clinician with ascitic fluid interpretation and with ascitic fluid characteristics in various diseases will increase the chances of controlling ascites early. The definite diagnosis of spontaneous bacterial peritonitis (SBP) is based on clinical evidence, neutrophil count and positive ascitic fluid culture. The objective of this study was to identify the bacterial agents from ascitic fluid and to determine their antibiotic resistance profile. Material and Methods: Aretrospective analysis of 132 ascitic fluid specimens collected for direct Gram's staining and culture during March 2012 to April 2013 was performed. The antibiotic susceptibility testing was done as per Clinical laboratory Standard Institute guidelines. The extended-spectrum beta-lactamase (ESBL) production in Gram negative isolates were determined. Results: On Gram's staining, microorganisms with pus cells were seen in 20.4% and culture was positive in 37.8% of samples. Most of the isolates were Gram negative (70%). Among Gram negative isolates, E. coli was most common isolate (74%) followed by Acinetobacter spp. (11.6%), Enterobacter spp.(5.8%), Pseudomonas aeruginosa (5.8%) and Klebsiella oxytoca (2.9%). Among Gram positive isolates, Staphylococcus aureus, Enterococcus faecalis and coagulase negative staphylococci were 73.3%, 20% and 6.7% respectively. ESBLs were detected among 17.6% of E. coli and 30.5% of Klebsiella oxytoca isolates and all were multidrug resistant. All the strains were found to be sensitive to imipenem. Interpretation & Conclusion: The knowledge of presumptive causative organism and their antibiotic resistance profile will help the clinician in choosing empirical antibiotic therapy in suspected patients of SBP.