Introduction: Coagulase-negative staphylococci (CONS) transformed from being commensals to pathogens causing a wide variety of infections. Meager nutritional requirements and ability to withstand various physical, chemical agents have made CONS a successful pathogen. Main virulence factor associated with CONS infections is biofilm formation. Biofilm helps CONS adhere to surfaces to escape the assault by immune mechanisms and antibiotics. The estimation of biofilm formation will help differentiate between commensal and pathogenic CONS. Purpose: To determine clinically significant CONS and to ascertain their virulence using qualitative and quantitative methods of biofilm detection. Materials and Methods: A total of 75 clinically significant isolates were taken up for the study. These isolates were segregated into two groups: Isolates with definite clinical significance (Group A 45 isolates) and isolates with moderate significance (Group B 30 isolates). Two qualitative methods Congo red agar method and tube method were employed. Quantitative detection of biofilm (adherence) was detected by microtiter plate (MTP) method. Results: The more sensitive and quantitative method was MTP method. In Group A, 20 were moderate biofilm producers and 14 were strong biofilm producers. In Group B, 8 out of 30 were moderate biofilm producers and 6 were strong biofilm producers. The comparison of the three methods showed that MTP method was more sensitive in detecting of biofilm and helps in quantitative assessment on the amount of biofilm formation. Statistical significance of the difference between Group A and Group B isolates was found to be statistically significant, P = 0.004. Conclusion: These methods are cost-effective and need minimal technical training. The detection of biofilm production will help differentiate pathogenic and commensal CONS. The reporting of biofilm will help the clinician to plan the appropriate line of therapy.