Clinical diagnosis of infectious diseases in Africa has been associated with increased misdiagnosis and mortality, but when laboratory testing is available, it remains underused. We retrospectively compared infectious diagnoses, test results, anti-microbial use, and patient cost with laboratory and physician surveys at a teaching hospital in Ghana to evaluate the potential barriers to laboratory use and financial impact for patients. Laboratory capacity was high, but physician survey results and objective data indicated a reliance on clinical judgment and empirical therapy. For the study period, 9-15% of malaria diagnoses, 34-43% of urinary tract infections (UTIs), and 62% of meningitis cases were supported by abnormal laboratory results. For the same period, 0.82-2.09 units of antibiotics were consumed per patient day, and patient cost for antibiotics was 4.8-21.6 times that of laboratory testing. Physician perception regarding the value of diagnostic testing is potentially a major barrier to laboratory use, resulting in empiricism, disproportionate anti-microbial administration, and cost to patients.