Infections caused by commensal organisms by changing to infectious life style generate much challenge to the current treatment strategies. Coagulase Negative Staphylococci (CoNS) are one of them, with their coexisting biofilm forming and multiple antibiotic resistance properties form important agents of nosocomial infection. To evaluate species distribution, biofilm formation, and antibiogram, CoNS isolates from various clinical samples were isolated. The presence of biofilm and associated genes icaAB, aap, atlE, embp, bhp, and fbe in CoNS was screened by PCR. The biofilm chemical composition and its correlation with the genotypes were also analysed. Staphylococcus epidermidis (59%) was found to be the most prevalent CoNS species. Most of the CoNS isolates harboring biofilm gene showed carbohydrate-protein-eDNA biofilm, whereas carbohydrate-protein biofilms were also observed. High percentage of multiple drug resistance, and biofilm gene frequency among these CoNS isolates point towards the need of periodic surveillance as CoNS are recently identified to cause difficult to treat infections.