In Nigeria, quinolones and β-lactam antibiotics are widely used to treat bacterial infections. This study aimed to identify the prevalence of resistance to these drugs and to determine the mechanisms of resistance to these agents. In total, 134 non-duplicate, Gram-negative enteric isolates of 13 species from different hospitals were investigated for susceptibility to a panel of antibiotics, carriage of plasmid-mediated quinolone and β-lactam resistance genes, production of extended-spectrum β-lactamases (ESBLs), and mutations within topoisomerase genes. The level of resistance to all antibiotics tested was extremely high, with minimum inhibitory concentrations for 90% of the organisms (MIC values) of ≥256μg/mL for all drugs. Of the 134 isolates, 92 had mutations within the quinolone resistance-determining region (QRDR) of or within and . In addition, the plasmid-mediated quinolone resistance genes , , and were identified. The allele, which has previously only been found in isolates from China, was identified in two isolates and one isolate. Of the 134 isolates, 23 (17.2%) carried , 11 (8.2%) carried a variant and 5 (3.7%) were positive for . Twenty-eight isolates (20.9%) produced ESBL variants, with a CTX-M variant being carried by 25 isolates (18.7%). In addition, six isolates (4.5%) carried variants [ACT-1 (1 isolate), DHA-1 (4 isolates) and CMY-2 (1 isolate)]. This study demonstrates a very high level of multidrug resistance amongst Gram-negative enteric bacilli isolated from different sites from patients in Nigerian hospitals as well as the presence of a variety of plasmid-associated resistance genes, including some identified from Africa for the first time.