Infections caused by aerobic gram negative bacilli (GNB) are common in hospitalized patients and result in serious infections such as bacteraemia and nosocomial pneumonia. Moreover, the emergence and spread of antimicrobial resistance among GNB represents a major problem. A variety of these GNB produce plasmid encoded beta lactamases mainly TEM and SHV enzymes. Mutations of the genes encoding these enzymes extend the spectrum activity to include extended-spectrum cephalosporins such as ceftazidime, cefotaxime and ceftriaxone and aztreonam. These enzymes are called extended spectrum beta lactamases (ESβLs). AmpC type beta lactamases also show activity against these antimicrobial agents.The aim of this study was to detect the rate of NIs caused by ESΒL producing GNB among neonates in the NICU of Manial Hospital, Faculty of Medicine, Cairo University and to control infection by tracing the source of infection and breaking the chain of transmission. Screening for B-lactamase production was done by oxoid stick method. Screening for ESβL production was done by antibiotic susceptibility disc diffusion method. Confirmation of ESβL production was done by combined disc method and Dade Behring MicroScan MIC detection method. Lastly plasmid profiles were done for isolated nosocomial GNB to trace the source of infection. In the present study, results showed that 72.3% of isolated organisms were GNB. 31.9% of isolated GNB were ESβL producers. ESβL producing GNB accounted for 23.1% of nosocomial isolates. Hands of Health Care Workers (HCWs) were a major source of transmission of infectious organisms from caregivers to patients. Hand washing is one of the most important factors in controlling the spread of bacteria and in preventing the development of infection.