PURPOSE: Cotrimoxazole preventive therapy (CPT) is a feasible, inexpensive, and well-tolerated way of using cotrimoxazole intervention for patients living with HIV/AIDS to reduce HIV/AIDS-related morbidities and mortalities caused by various bacteria, fungi, and protozoa. The aim of this study was to evaluate the use of cotrimoxazole as a prophylaxis therapy among patients living with HIV/AIDS at Gondar University Referral Hospital (GURH), northwestern Ethiopia. MATERIALS AND METHODS: A retrospective cross-sectional study was used to evaluate the use of cotrimoxazole as a prophylaxis therapy among people living with HIV/AIDS at GURH, northwestern Ethiopia from September 2013 to October 2015. Medical records of 264 patients were selected by using systematic random sampling technique from the sampling frame list of all patients' medical records. Data were collected from patients' medical records using the structured checklist and evaluated against World Health Organization (WHO) guidelines on the use of cotrimoxazole prophylaxis. The quantitative data were analyzed using the statistical packages for social sciences Version 20. Descriptive and binary logistic regression analyses were used to describe and assess the association between different variables. RESULTS: Approximately 95 (36.0%) patients were at WHO clinical stage III at the start of CPT. The use of CPT was consistent with the guidelines in the rationale for indication 200 (75.75%) and dose 263 (99.62%), despite the presence of contraindications in 24 (9.90%) patients. The occurrence of cotrimoxazole-associated side effects was higher in the first month of therapy. Problems regarding drug-drug interactions were identified in 63 (23.86%) patients, and 92 (34.84%) patients discontinued CPT due to different reasons. CONCLUSION: Although the practice of discontinuation of CPT and follow-up for adverse drug effects were not consistent with WHO guidelines on the rational use of cotrimoxazole prophylaxis, the use of CPT among people living with HIV/AIDS at GURH was appropriate. Health professionals who were working in the antiretroviral therapy units should update themselves and adhere to the available updated guidelines to reduce the occurrence of adverse effects and prophylaxis failure.