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Introduction: Biofilms represent a complex structure comprising prokaryotic cells, proteins, sugars, and DNA as major constituents. Bacteria can grow in biofilms and associated with human infections and considered to be highly resistant to antibiotics. There are various methods to detect biofilm production like tissue culture plate (TCP), tube method (TM), and Congo red agar method (CRA). Objectives: This study aimed to compare three methods for the detection of biofilm formation in the clinical isolates Staphylococcus aureus and Escherichia coli. Methods: A total of 60 clinical isolates of S. aureus and E. coli were subjected to biofilm detection methods by TM, CRA and TCP. Results: Out of the total 60 clinical isolates of S. aureus and E. coli, TCP method detected 33 (55%) as strong, 15 (25%) as moderate and 12 (20%) as weak/non-biofilm producers. The sensitivity of TM and CRA was 56.3% and 60.4%, and the specificity was 58.3% and 66.7% respectively. In our validation of the diagnostic biofilm production tests, the TCP method was superior to TM and CRA methods for biofilm detection. Conclusion: The study concluded that the TCP method is accurate and reliable method for the detection of biofilm formation in the clinical isolates S. aureus and E. coli compared to TM and CRA methods, and can be recommended as a general screening method for the detection of biofilm producing bacteria in clinical laboratories. [ABSTRACT FROM AUTHOR]