The purpose of the study was to determine extended spectrum beta-lactamase (ESBL) activity in nosocomial Gram-negative bacilli. The nosocomial bacterial isolates from Dicle University Hospital Infectious Disease Laboratory and Central Laboratory during a one-year period were assessed according to Centers for Disease Control (CDC) criteria. In this period, 124 nosocomial Gram-negative bacteria were studied to determine extended spectrum beta-lactamase (ESBL) expression by using E-test method. The ESBL expression was seen mostly in 45% (15/33) of Klebsiella spp, 32% (10/31) of Escherichia coli %27 (3/11) of Pseudomonas spp., 27% (3/11) of Enterobacter spp., 22% (2/9) of Acinetobacter spp. and 14% (1/7) of Proteus spp.. Totally, ESBL expression was detected in 38 out of 124 strains (31%). Extended spectrum beta-lactamase production should be screened in nosocomial Gram-negative bacteria especially in E. coli and Klebsiella spp. The presence of ESBL must be considered for choosing appropriate antibiotic treatment of their infections.