Background: Kingella kingae and Neisseria meningitides are gram-negative bacteria, causing several life-threatening diseases and considered as opportunistic pathogens in the upper respiratory tract of healthy carriers. The detection of these both bacteria species is difficult in routine culture methods. Objective: This study aimed to find the occurrence rate of K. kingae and N. meningitides colonizing upper respiratory tract of young Jordanian children, and to determine the antimicrobial susceptibility profile of the isolates. Methods: A total of 300 samples of throat and nasal swabs were collected from out- patients Jordanian children aged between 6 months and 5 years, who were admitted to Pediatrics' clinics department at the Jordan University Hospital and Al-Bashir Hospital over the period October 2018 through January 2019. Samples were cultured for detection K. kingae and Neisseria species including specially N. meningitides. Their suspected growth was identified and tested using microbiology culture methods and polymerase chain reaction (PCR) method. Additionally, DNA was extracted directly from one 100 samples and was investigated only for K. kingae using real- time PCR assay. Results: This study showed the absence of K. kingae in all cultured samples, while Neisseriaspecies was detected in 21 (7 %)including one N. meningitides isolate(0.3%). The results of antibiotic susceptibility testing indicated presence of few percentage of Neisseria species isolates resistant 100 % to clindamycin, oxacillin and vancomycin, whereas all were susceptible for chloramphenicol (100%)levofloxacin and gentamycin , and less to ampicillin(90.6%) and erytromycin ( 85.7%), respectively. Conclusion: This study shows the absence of K. kingae and the rare occurrence of N. meningitides colonizing the upper respiratory tract of young Jordanian children over the 4-month period of study.