Background: The detection of extended-spectrum β-lactamase-producing (ESBL) bacteria is of importance for infection control and epidemiological surveillance. Objective: The purpose of the present study was to compare two phenotypic methods for the detection of ESBL-positive Enterobacteriaceae and Pseudomonas species. Methodology: This cross sectional study was carried out in the Department of Microbiology at Mymensingh Medical College, Bangladesh from January 2011 to June 2011. Patients of all ages and genders presented with UTI or wound infection were taken as study population. Gram negative bacilli (GNB) were analyzed by two methods used for routine susceptibility testing which were Disk diffusion methods and MIC reduction methods. Two methods designed for the detection of ESBL production Ceftazidime and Ceftazidime plus Clavulinic acid, (CAZ/CAZC) and Cefotaxime and Cefotaxime plus Clavulinic Acid (CTX/CTXC) were used and the PCR was considered as gold standard for evaluation of the other test methods. Result: A total number of 300 GNB were isolated and identified of which 214(71%) were ESBL positive. For the disk diffusion method, resistant to third generation Cephalosporins were the highest 87.0%, when tested by ceftazidime and by MIC reduction methods were 67%. For the phenotypic confirmatory methods, specificities were 64% by (CAZ/CAZC), 59% by (CTX/CTXC) and by both method 52%. Among the phenotypic confirmatory ESBL positive strains by Genotypic method ESBL positive were 50.46% TEM, 18.69 % SHV and 46.72% CTX-M gene. Conclusion: Two-step strategies using both DDDT phenotypic methods are useful diagnostic tools for the detection of ESBL from the Gram negative bacilli.