Miscellaneous Gram negative bacteria are seriously affecting broiler chickens and poultry industry in Egypt. The present study aimed to isolate and completely identify oxidase positive Gram negative bacterial pathogens recovered from different pathological lesions in broiler chickens by conventional biochemical tests, API20NE and microbact24e system. Samples were taken from 200 Hubbard and Ross broiler chickens of different ages (3-5weeks), from different farms in Beni-Suef and El-Fayoum Governorates during the period from January 2016 to April 2016.  Bacteriological examination showed that Gram negative bacteria were 165 (82.5%) of isolates of which 105 isolates (52.5%) were oxidase positive while 60 isolates (30%) were oxidase negative. The most prevalent bacterial isolates were Pseudomonas aeruginosa (43 isolates), Aeromonas hydrophila (35 isolates), Pasteurella gallicida (12 isolates), Plesiomonas shigelloides (10 isolates), and Vibrio vulnificus (5 isolates) with incidences of 21.5%, 17.5%, 6% 5%, and 2.5%, respectively.