Antimicrobial resistance is a global challenge requiring reliable surveillance data collection and use. Prior studies on resistance in Zambia depended on laboratory methods with limited standardization. Since 2015, the University Teaching Hospital (UTH) microbiology laboratory has used the Vitek 2 Compact (bioMerieux, Inc., Marcy-l’Étoile, France) for standardized identification and susceptibility testing. We conducted a cross-sectional study of 2019 bacterial isolates collected from July 2015 to April 2017 to identify bacterial causes of infections, their susceptibility to commonly used antibiotics at UTH, and develop hospital antibiograms with a multidisciplinary team using World Health Organization guidance. We found high levels of antibiotic resistance among Gram negative bacteria. Escherichia coli and Klebsiella pneumoniae were highly resistant to all antibiotics except amikacin and carbapenems. E. coli had susceptibilities of 42.4% to amoxicillin/clavulanic acid, 41.4% to ceftriaxone, 40.2% to ciprofloxacin, and 10.4% to trimethoprim/sulfamethoxazole (TMP/SMX). K. pneumoniae had susceptibilities of 20.7% to amoxicillin/clavulanic acid, 15.6% to ceftriaxone, 48.5% to ciprofloxacin, and 12.3% to TMP/SMX. The high resistance to 3rd generation cephalosporins indicates high rates of beta-lactamase production. This is information that clinicians need to inform clinical decision making and choice of empiric antibiotics and that UTH requires to inform antimicrobial stewardship such as improvements in antibiotic use.