BACKGROUND: Body fluids like ascitic fluids, pleural fluids, cerebrospinal fluids (CSF) etc. are sent for culture in a clinical microbiology laboratory to achieve etiological diagnosis. However the yield of such cultures is usually very low. So, ongoing monitoring of prevalent pathogenic organisms and their sensitivities help the clinicians institute therapy in absence of a culture report. AIMS: The study was done to identify the common pathogens isolated from body fluids along with their antimicrobial susceptibility pattern and also to evaluate the impact of enrichment on their culture positivity. SETTING AND DESIGN: A 3-month prospective analytical study was done in a tertiary care hospital. MATERIALS AND METHODS: A total of 333 Body fluids were processed; 103 of them were ascitic fluids, 71 pleural fluids, 139 CSF and 20 other fluids. They were processed by plating the direct sample and after enrichment. Enrichment was done by two methods: in SoyabeanCaesin digest broth (274 samples) and by BACTEC (59 samples).Isolates were identified by routine procedures & their antimicrobial susceptibility determined as per CLSI guidelines. The results were analyzed using Microsoft Excel® software using p<0.05 as the cut-off for significance. RESULTS: Gram negative isolate were obtained from 21.3% of the samples. The common isolates were Pseudomonas (20.7%), Acinetobacter (11.6%), Citrobacter (10.7%) and E. coli (10.7%). The antibiotics most effective against Gram negative pathogens were Gentamicin (47.5%), PipercillinTazobactam (51.6%), Amikacin (56.7%) and Cefoperazone-Sulbactam(65.3%). Gram positive isolates, obtained from 9% of the samples, mostly consisted of MSSA, Enterococcus and CONS, for which Ciprofloxacin (48%) followed by Cotrimoxazole (40%) and Erythromycin (28.6%) showed reasonable efficacy. The Culture positivity with direct plating, Soyabean-Caesin broth enrichment and BACTEC was 14.41%, 29.19% and 42.37% respectively. Increase in positivity by Soyabean-Casein broth was maximum for pleural fluids (12%) followed by ascitic fluids (11.6%) and CSF (11.52%).Using automated system the corresponding increases were 20.7%for ascitic fluids and 5.4%for pleural fluids. The mean time for identification using direct plating, enrichment method and BACTEC were 48 hours, 72 hours and 40 hours respectively. CONCLUSION: Gram negative isolates are commonly isolated pathogens from body fluids in our setup. Enrichment of body fluids improved yield of pathogens. In resource-poor settings simple enrichment in blood culture bottles can increase culture positivity of these precious samples.