Biofilm producer bacteria cause nosocomial, chronic, and recurrent human infections. It is hard to treat biofilm-embedded bacteria because they are more resistant to antimicrobials than planktonic bacteria. The present study aimed to investigate different methods for detecting biofilms in the clinical isolates of Escherichia coli and Pseudomonas aeruginosa. The study was carried out at the Department of Microbiology, Ardabil Branch, Islamic Azad University, Ardabil, Iran, from January 2019 to June 2019. A total of 320 clinical samples were collected from educational and medical centers in Tehran, Iran which from them 100 isolates of E. coli and P. aeruginosa were identified by standard microbiological procedures and subjected to biofilm detection methods. Biofilm detection was tested by Congo red agar (CRA), tube method (TM), microtiter plate assay (MTPA). The MTPA was considered to be superior to CRA and tube TM. From the total of 100 clinical isolates, MTPA detected 38 (38%) isolates as biofilm-positive phenotype, of which 30 as strong, and 8 as moderate biofilm-forming isolates. It can be concluded from the current study that the MTPA is a more quantitative and dependable assay for the detection of biofilm-forming microorganisms as compared to other methods, and it can be recommended as a general screening method for the detection of biofilm-producing bacteria in laboratories.