In Gabon, few data exist on extended-spectrum beta-lactamases-producing Enterobacteriaceae (ESBL-PE). This study investigated ESBL-PE prevalence and the associated resistance genes in clinical samples (n = 5,956) and anal swabs (n = 78) analyzed in eight hospitals and a medical analysis laboratory in Gabon from January 2016 to March 2018. Matrix-Assisted Laser Desorption Ionization-Time Of Flight (MALDI-TOF) mass spectrometry analysis identified 790 Enterobacteriaceae isolates (n = 712 clinical samples and n = 78 fecal samples). ESBL-PE prevalence (Müller-Hinton agar disk diffusion method and double-disk synergy test) was 11.8% (84/712) in clinical samples (15.5% from inpatients and 7.1% from outpatients; p < 0.05) and 16.7% (13/78) in carriage isolates. Most ESBL-PE were isolated from urine samples (46/84). In clinical and carriage ESBL-PE isolates, Escherichia coli was predominant (42.8% and 61.5%; phylogroups A, B1, B2, and D), followed by Klebsiella pneumoniae (41.7% and 23.1%). Multiplex PCR and bi-directional sequencing showed that CTX-M group 1 (blaCTX-M-15) was predominant in clinical and carriage ESBL-PE (94% and 92.3%) among which 85.7% and 92.3% also harbored one to three β-lactamase-encoding genes (blaTEM-1, blaOXA-1, or blaSHV-1). Resistance genes were detected in all hospitals in Gabon. ESBL-PE prevalence in Gabon has not reached alarming levels yet, but corrective and monitoring measures are needed to curb their emergence.