Infections with bacteria producing extended-spectrum beta-lactamases (ESBLs) are increasing across Africa. This study reports on ESBL-producing Enterobacteriaceae as significant causes of infections and antibiotic resistance at Korle-Bu Teaching Hospital in Accra, Ghana. Of 300 isolates examined, 49.3% produced ESBLs. The prevalence of ESBLs was significantly high among isolates from neonates (28 of 43, 65.1%; relative risk = 1.62, 95% confidence interval = 1.33-2.13, P = 0.002) and adult patients > 65 years of age (36 of 51, 70.5%; relative risk = 1.89, 95% confidence interval = 1.41-2.40, P = 0.001). A marked increase in minimum inhibitory concentrations of ESBL-positive species was noticed compared with those for the other strains. Using these concentrations, we found that 26 (17%) ESBL producers were resistant to two or more antibiotics (aminoglycosides, fluoroquinolones, sulfonamide, and carbapenems) whereas 5 (3.2%) non-ESBL producers were multidrug resistant. Regular ESBL detection and evaluation of antibiotic resistance may help reduce the spread of ESBLs and antibiotic resistance in Ghana.