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Resistance to antibiotics is increasing globally and is a threat to public health. Research has demonstrated a correlation between antibiotic use and resistance development. Developing countries are the most affected by resistance because of high infectious disease burden, limited access to quality assured antibiotics and more optimal drugs and poor antibiotic use practices. The appropriate use of antibiotics to slow the pace of resistance development is crucial. The study retrospectively assessed antibiotic prescription practices in four public and private primary health-care facilities in Eastern Region, Ghana using the WHO/International Network for the Rational Use of Drugs rational drug use indicators. Using a systematic sampling procedure, 400 prescriptions were selected per facility for the period April 2010 to March 2011. Rational drug use indicators were assessed in the descriptive analysis and logistic regression was used to explore for predictors of antibiotic prescription. Average number of medicines prescribed per encounter was 4.01, and 59.9% of prescriptions had antibiotics whilst 24.2% had injections. In total, 79.2% and 88.1% of prescribed medicines were generics and from the national essential medicine list, respectively. In the multivariate analysis, health facility type (odds ratio [OR] = 2.05; 95% confidence interval [CI]: 1.42, 2.95), patient age (OR = 0.97; 95% CI: 0.97, 0.98), number of medicines on a prescription (OR = 1.85; 95% CI: 1.63, 2.10) and ‘no malaria drug’ on prescription (OR = 5.05; 95% CI: 2.08, 12.25) were associated with an antibiotic prescription. A diagnosis of upper respiratory tract infection was positively associated with antibiotic use. The level of antibiotic use varied depending on the health facility type and was generally high compared with the national average estimated in 2008. Interventions that reduce diagnostic uncertainty in illness management should be considered. The National Health Insurance Scheme, as the main purchaser of health services in Ghana, offers an opportunity that should be exploited to introduce policies in support of rational drug use.