Extended Spectrum β-lactamase (ESBL)-producing Enterobacteriaceae are of major concern as they are implicated in multidrug resistant nosocomial infections. They are listed on a recently published global priority list of antibiotic-resistant bacteria by the World Health Organization which raises concern in both healthcare and community settings. This study aimed at determining the frequency of ESBL genes in multidrug resistant human clinical Enterobacteriaceae isolates from Edo state Nigeria and to characterize the resistance mechanisms using whole genome sequencing. A total of 217 consecutive clinical isolates of Enterobacteriaceae, selection based on inclusion criteria, were collected from March-May 2015 from three medical microbiology laboratories of hospitals in Edo state Nigeria. All isolates were analyzed using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Antibiotic susceptibility testing was performed by Kirby-Bauer method and minimum inhibitory concentration (MIC) determination by E-test method. Double disc synergy test was used to screen for the production of ESBL. Whole genome sequencing (WGS) was performed for isolate characterization and identification of resistance determinants. Out of 217 consecutive clinical Enterobacteriaceae isolates, 148 (68.2%) were multi-drug resistant. Of these multi-drug resistant isolates, 60 (40.5%) were positive for the ESBL phenotypic test and carried ESBL genes. CTX-M-15 was the predominant ESBL found, among 93.3% (n = 56/60). Thirty-two plasmid incompatibility groups and 28 known and two new sequence types were identified among the ESBL isolates. The high occurrence of CTX-M-15 with associated resistant determinants in multidrug resistant Enterobacteriaceae harboring different plasmid incompatibility groups and sequence types calls for the need of continuous monitoring of this resistance threat to reduce its public health impact. To our knowledge, this study presents the first genomic characterization of ESBL production mediated by blaCTX-M-15 in human clinical isolates of Enterobacter hormaechei, Citrobacter werkmanii and Atlantibacter hermannii from Nigeria.