Introduction: Coagulase negative staphylococci (CoNS) are the most frequent blood culture isolates and an important cause of nosocomial blood stream infections especially in catheterised patients. CoNS are also the most common contaminants of blood cultures and are proven to be especially problematic. These uncertainties may result in over diagnosis and indirect overuse of antimicrobials especially vancomycin. Aim: To determine the clinical significance of CoNS isolated from blood culture of patients admitted in a tertiary care hospital. Materials and Methods: A prospective cross-sectional study was conducted in Bharati Hospital Research Center, Pune, Maharashtra, India, from August 2019 to July 2020 (One year). Patients whose paired/multiple blood culture samples showed pure growth of CoNS were included in the study. CoNS were identified by Vitek 2 system and antimicrobial susceptibility was reported. Clinical history of all patients was taken who showed pure growth of CoNS in either one or multiple sites of the automated blood culture bottles. Isolated CoNS were considered as pathogen only if clinical and laboratory parameters are fulfilled. Chi-square test was used to find out statistical significance of isolated pathogenic CoNS. Results: A total 147 CoNS isolated from blood cultures of suspected patients of sepsis were included in study. About 23 (15.6%) CoNS were isolated from both the sites. Remaining 124 (84.4%) CoNS were isolated from single site. CoNS were considered as pathogen in all 23 cases based on clinical and laboratory criteria. The most frequent isolated CoNS was S. haemolyticus 7 (30.4%), followed by S. epidermidis 5 (21.7%), S. hominis 3 (13.1%) , S. lugdunensis 3 (13.1%), S. scuiri 2 (8.7%), S. xylosus 1 (4.3%), S. caprae 1 (4.3%), S. capitis 1 (4.3%). Methicillin resistance was observed in 15 (65.2%) CoNS strains. Conclusion: Patients suffering from CoNS from their blood stream should be carefully evaluated clinically. Proper blood collection techniques need to be followed to avoid contamination of blood culture samples and to avoid diagnostic dilemma.