Strict adherence to anti-retroviral therapy (ART) is critical for sustained suppression of viral replication and prevention of opportunistic infections that often lead to disability or death. With increased availability of ART in recent years, achievement of optimal adherence has become very important in the management of HIV/AIDS in Eritrea. To assess the factors affecting adherence to ART of HIV-positive patients visiting ART clinics in Halibet national referral hospital and Hazhaz hospital, a cross-sectional descriptive study was conducted from January to March 2016. A simple random sampling technique was used to select the study areas. Participants were selected using systematic random sampling from patients who came to take their medication from the ART out-patient department of the hospitals. Semi-structured interviews were conducted with 150 patients. Almost half of the patients were between the age group of 30-40 years. Patients with an educational level of 9-12th grade had higher adherence levels. There was a significant association between age of participants and the dose missed (P= 0.002, 95% CI). The presence of adverse drug reaction (ADRs) was common, and 65% of the participants experienced them. 78.5% of the patients who experienced ADRs reported they would consult a doctor when experiencing the symptoms. 56.6% of the participants said that they always take ART on time. 62% reported they did not miss any dose of anti-HIV medication in the last seven days. Overall, the patients had good adherence to ART treatment. Perceived stigma and discrimination and medication side effects were the main reasons for non-adherence. Continuous health education on ART management and periodic counseling emerged as contributors for a better adherence level.