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Background: Opportunistic infections are diseases that cause infections in people whose immune systems are weakened. It is the most public health problem and the leading cause of morbidity and mortality of AIDS-related infections in developing countries. Identification of opportunistic infections is the core for developing a specific intervention. Despite this fact, information about the burden of opportunistic infections is lacking in the particular study. Therefore, this study aimed to assess the prevalence and associated factors of opportunistic infections among HIV-infected patients in Eastern zone of Tigray, northern Ethiopia. Methods: An institutional-based cross-sectional study was done among HIV/AIDS patients taking antiretroviral therapy in Northern Ethiopia. A total of 394 study subjects was selected using a systematic random sampling method. Data was collected using a structured questionnaire. The collected data was entered and analyzed by SPSS version 22. Bivariate and multivariable logistic regression analysis with 95% confidence interval were used to find factors associated with opportunistic infections. The adjusted odds ratio was calculated to show the strength of the association. Variables with p-value of < 0.05 were considered statically significant. Results: The mean age of the respondents was 41 (± 10). Of the study participants, about 52% (95% CI: 47.5-57.1) developed opportunistic infections. Being housewife occupation (AOR = 0.17; 95% CI: 0.07-0.46), CD4+ count < 200 cells/µl (AOR = 2.38; 95% CI: 1.3-4.67), poor adherence (AOR = 4; 95% CI: 1.95-8.2), and bedridden functional status (AOR = 13.9; 95% CI: 5.2-37) were independently associated with opportunistic infections. Conclusion: This study stated the prevalence of opportunistic infections among HIV-infected patients is high. Being housewife occupation, poor ART adherence, low CD4+ count, and bedridden functional status were found to be associated with development of opportunistic infections. Therefore, health care providers must give continuous health education on primary prevention of opportunistic infections.