Background and Objectives: Obligate anaerobic bacteria are known to cause various infections in human beings. We aimed to determine the prevalence and spectrum of obligate anaerobes encountered in pus aspirate, sterile fluids and tissue samples received for routine bacterial culture and sensitivity. Materials and Methods: A total of 160 samples including tissue, sterile body fluids and pus aspirate were collected , ana- lysed for prevalence and spectrum of obligate anaerobes. Identification of obligate and facultative anaerobes was done by automated MALDI-TOF and Vitek 2 method. Results: Among 160 samples, 75 samples (46.8%) yielded obligate anaerobes out of which 41 samples (26%) yielded obli- gate anaerobes along with facultative anaerobes which was significant (p value=0.031) and 34 samples (21%) yielded only obligate anaerobes. 90 obligate anaerobes were isolated from 75 samples among which only 34 (37.7%) samples yielded only obligate anaerobes and 56 (62.2%) yielded both obligate and facultative anaerobes. Gram stain with polymicrobial appear- ance (p value 0.02) was found to be significantly associated with growth of obligate anaerobes. Clinical conditions where obligate anaerobes were commonly associated were appendicular abscess, empyema, fournier’s gangrene, diabetic foot, ludwigs angina and deep abscess. Out of 75 positive samples 30 (40%) patients had predisposing conditions like diabetes mellitus, hypertension etc. Total of 90 obligate anaerobes and 49 facultative anaerobes were isolated. The common obligate anaerobes were Bacteroides fragilis 18 (20%), Prevotella spp. 20 (22.2%), and Clostridium spp. 8 (8.88%). Facultative anaerobes like Escherichia coli 25 (34.7%), Klebsiella species 15 (20.8%), Enterococcus faecalis 19 (26.3%) were isolated. Antibiotic sensitivity was performed for facultative anaerobes by Kirby bauer disc diffusion method. Out of 15 Escherichia coli isolates resistance was commonly seen for ampicillin 13 (86.6%), cephalosporins 11 (73.3%), ciprofloxacin 10 (66.6%) and Piperacillin tazobactum 8 (53.3%). In Klebsiella species resistance were commonly seen to Ampicillin 6 (100%), ceph- alosporins 2 (33.3%) and ciprofloxacin 2 (33.3%). Conclusion: There was significant isolation of obligate anaerobes along with facultative anaerobes in clinical samples re- ceived for aerobic culture and sensitivity. There is a need for isolation of these bacteria routinely and a scope for doing anti- biotic susceptibility testing, which will help in evidence-based medicine and a better clinical outcome by giving appropriate therapy.