Introduction: Staphylococci are major nosocomial pathogen associated with indwelling medical devices, especially with intravascular catheter (I V) related infections. Major virulence factor of Staphylococcus is its ability to form biofilm on polymeric surfaces. Objective: __ampersandsignbull; To isolate and identify the pathogens colonising IV catheters. __ampersandsignbull; To select the Staphylococcus species and to detect their ability to produce biofilms along with antimicrobial susceptibility testing. Material and methods: A total of 373 IV catheter tips from 373 patients were collected and processed for isolation of bacterial pathogen. Total 119(31.9%) IV catheter tips were culture positive. 100 Staphylococcus strains were tested for biofilm detection by Tissue culture plate (TCP) method and Tube method (TM). Antimicrobial susceptibility testing was done by Kirby-Bauer disc diffusion method as per CLSI guidelines. The time frame since insertion and removal of catheter was also noted. Results: 125 organisms were isolated from 119(31.9%) culture positive IV catheter tips. Staphylococcus spp.(80%) was the most common organism followed by Enterococcus spp(9% ). 93(24.4%) catheters were colonised within one week of insertion.56% of the Staphylococci were Coagulase negative staphylococci (CoNS) and 44% were S.aureus. Of 100 Staphylococcus spp. 84% were biofilm producer by TCP method and 75% by Tube method. Sensitivity and specificity of TM method vis-a-vis TCP method was 89.3%and 100% respectively. Conclusion: Majority of Staphylococcus isolated from IV catheters were biofilm producers. TCP method is standard method for detection of strong, moderate and weak biofilm producing strains, but TM method is technically simple. Antimicrobial resistance was significantly higher in biofilm producing Staphylococcal species.