Extended spectrum β-lactamases (ESBLs) have emerged as important mechanisms of antimicrobial resistance to beta-lactam drugs in hospitals, thereby limiting the available treatment options for infections caused by these microorganisms. The objectives of this study were to determine the prevalence of ESBL production among clinical isolates of Gram-negative bacilli from Obafemi Awolowo University Teaching Hospitals Complex, Ile-Ife, Nigeria as part of the global efforts to provide information for the containment of the spread of antimicrobial resistance. This is a laboratory-based study of 186 consecutive non-duplicate Gram-negative bacilli (GNB) isolated from diverse clinical samples at the Microbiology laboratory of the hospital after standard aerobic cultures. The isolates were identified by conventional biochemical test scheme and commercial GNB API 20E identification kit. Antimicrobial susceptibility testing (AST) of each isolate was determined by the disk diffusion technique. Isolates presumptively identified as ESBL producers from the AST assay were confirmed by the combination disks method. Escherichia coli (n=43; 23.1%) predominated among the organisms, followed by Enterobacter spp (n=23; 12.4%) and Klebsiella spp (n=22; 11.8%). Most of the organisms (95.7%) were resistant to tetracycline and ampicillin, but the least resistance (4.3%) was exhibited to imipenem. Fifty-eight (31.2%) isolates were ESBL producers with a preponderance of E. coli (n=15; 25.9%). The ESBL producers were commonly isolated from urine samples (n=31; 53.4%), and were significantly more resistant to levofloxacin, ciprofloxacin, nalidixic acid, gentamicin, augmentin, cefepime, ceftriaxone, ceftazidime and cefotaxime (p<0.05). This study has highlighted the therapeutic implications of the presence of EBSL in clinical isolates which was depicted by their multi-drug resistance phenotypes, thus emphasizing the need for continuous surveillance of bacterial resistance, institution of antimicrobial resistance stewardship in our hospital and regular screening of ESBLs in clinical isolates to prevent treatment failure.