Infection of sterile body fluids could be life-threatening. Monitoring of prevailing infectious agents and their resistance pattern through antibiogram helps in appropriate selection of antibiotics and strengthens antibiotic stewardship. The aim of this study is to isolate infective bacteria from sterile body fluids and to determine antibiotic resistant pattern of these isolates. This retrospective observational study was conducted through January 2018–December 2018 in a tertiary care hospital at central Delhi. Sterile body fluids were processed as per the standard laboratory procedures. Antibiotic susceptibility pattern was determined by Kirby Bauer’s disk diffusion method and interpreted as per Clinical and Laboratory Standards Institute guidelines. A total of 3,703 fluid samples were studied. Out of them 49.9% were ascitic fluid, 39.4% were pleural fluid, and 10.7% were others such as pericardial fluid, synovial fluid, bile, peritoneal dialysis fluid, and bronchoalveolar lavage fluid. Bacterial isolation rate was 12.93%. Predominant organisms were Escherichia coli (31%), Acinetobacter spp. (20%), Klebsiella spp. (16%), Staphylococcus aureus (11%), and Enterococcus spp. (10%). Isolation of vancomycin resistant enterococcus was 9.5% and Methicillin resistant S. aureus was 15.6%. Gram-negative isolates were 100% sensitive to colistin, followed by imipenem and gentamicin. Increased resistance shown toward third generation cephalosporins, amoxicillin + clavulinic acid, and piperacillin + tazobactum. Klebsiella spp. was the most resistant isolate, while Enterococcus spp. was the most resistant among gram positive isolates. Changing antimicrobial pattern poses challenge in treating infective agents. This culture antibiogram helps in appropriate selection of antibiotics in our setting and eventually decrease antibiotic resistant and patient’s morbidity and mortality.