Background Biofilms are defined as microbially derived sessile communities characterized by the cells to the substratum or to each other irreversibly attached. They are embedded in matrix of extracellular polymeric substances (EPS) which they have produced and exhibit an altered phenotype with respect to growth rate gene and gene transcription . Biofilm are associated with many medical conditions including indwelling medical condition devices, dental plaque, upper respiratory tract,infections,peritonitis and urogenital infection. Objectives: Compare and evaluate in-vitro methods: tissue culture method and tube method for the detection of biofilm formation by Staphylococcus aureus isolates from various clinical samples. Material and methods: The present study was carried out from January 2023 to December 2024 in the Department of Microbiology, Jawaharlal Nehru Medical College, KAHER Belagavi. A total of 145 Staphylococcus aureus isolates which were isolated from various clinical isolates received in the Department of Microbiology, Jawaharlal Nehru Medical College at, KAHER, received from KLE Dr. Prabhakar Kore Hospital, Belagavi. Result: A total of 145 Staphylococcus aureus isolates were tested for biofilm production by Tissue culture plate (TCP) and Tube method (TM). Tissue culture plate method was considered as gold standard which showed 25(17%) of Staphylococcus aureus isolates to be strong biofilm producers and 53(36%) as moderate biofilm producers, and 67(46%) as none or weak biofilm producers. Tube method showed 17(12%) of Staphylococcus aureus isolates to be strong biofilm producers, 47(32%) as moderate biofilm producers were and 81(55%) none or weak biofilm producers. Conclusion: This study showed Tissue culture plate method to be more reliable method to detect biofilm production from Staphylococcus aureus isolates .