Aims: The aim of this work was to determine the resistance profile and to investigate the production of extended spectrum β-lactamases (ESBL) by clinically relevant Gram-negative Bacillus (GNB) strains. Methodology: About 191 strains were isolated from 1823 samples collected at the HKM National Hospital and University Center of Cotonou (Benin). Species identification was done with the Api 20th gallery. Two methods were used to search for β-lactamase production: the liquid acidimetric test for penicillinases and double halo method for ESBL. The susceptibility to conventional antibiotic molecules was investigated by the disk diffusion method. Polymerase Chain Reaction (PCR) was used to identify blaTEM and blaSHV genes in the β-lactamases. Results: A prevalence of 10.48% of GNB was recorded. Among the isolated strains, 51.31% came from samples collected from in-patients and 48.69% from out-patients’ samples. The most contaminated samples were urine (43.98%), pus (34.58%) and blood (9.42%). Majority of the isolated species included: Klebsiella pneumoniae (28.27%), Acinetobacter spp. (18.32%), Pseudomonas aeruginosa (15.72%), Escherichia coli (14.15%) and Enterobacter cloacae (12.04%). More than the half (57.07%) of the strains produced penicillinases; whereas 16.76% were ESBL-producers and these occurred only among Klebsiella pneumoniae, Enterobacter cloacae, Escherichia coli and Enterobacter agglomerans. The ESBL-producing strains were cross-resistant to beta-lactams. Imipenem is the most effective antibiotic on all isolated strains. ESBL-producing GNB strains possessed both the blaTEM gene and the blaSHV gene in a proportion of 25%; 37.5% of the strains had only the blaTEM gene and 12.5% of the strains had only the blaSHV gene. Conclusion: ESBL-producing strains of K. pneumonia in the hospital environment were the major carriers of blaTEM and blaSHV. Given this situation, it is necessary to continue research to identify resistance genes.