Our objective was to evaluate the performance of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for identification of the Chryseobacterium and Elizabethkingia spp. isolated from pediatric patients at Hacettepe University Hospital using 16S rRNA gene sequencing as the gold standard and to determine the antimicrobial susceptibility patterns of the isolates and baseline characteristics of patients. All stored Chryseobacterium and Elizabethkingia spp. isolated from various clinical specimens (sputum, blood, and urine) of pediatric patients at Hacettepe University Hospital between 2012 and 2016 were included in this study. Minimum inhibitory concentrations of 10 antimicrobial agents were determined by Etest for all isolates. To determine the baseline characteristics of patients, medical records of all patients were retrospectively reviewed. In total, 18 isolates of Chryseobacterium spp. (16 C. indologenes, 2 C. gleum) and 5 isolates of Elizabethkingia spp. (3 E. meningoseptica, 2 E. anophelis) were identified by 16S rRNA sequencing. MALDI-TOF MS correctly identified 19 (82.6%) isolates to the species level. The quinolones (ciprofloxacin and levofloxacin), trimethoprim/sulfamethoxazole and piperacillin/tazobactam showed the highest spectrum of activity against the overall collection of isolates. Cystic fibrosis (CF) was the underlying disease in 81.8% of patients. To our knowledge, this study includes the largest number of Chryseobacterium spp. isolated from clinical specimens of pediatric patients in Turkey. In this study, we also report the first clinical isolate of E. anophelis in Turkey. Since, the majority of strains were isolated from patients with CF; larger, prospective clinical studies are needed to establish whether chryseobacteria could be considered as an emerging opportunistic pathogen in patients with CF.