Background Viral suppression among people living with HIV is the main goal of antiretroviral therapy (ART). Despite the treatment, more than four million people and over 55 thousand patients globally and in Ethiopia had virological failure respectively in 2017. The purpose of this study was to identify determinants of virological failure among patients on ART in central Oromia, Ethiopia. Methods A facility-based unmatched case–control study was conducted. Sample size was calculated using EpiInfo software, and 504 randomly selected patient’s records were analyzed. Case to control ratio was one to one. Separate sampling frames were prepared for cases and controls using medical registration numbers (MRN) from patients’ registrations data. Cases and controls were selected from respective sampling frames. Respective patient charts were traced using the MRN, and data were extracted from patient charts using data abstraction format. The data were entered using EpiInfo version seven and analyzed using SPSS version 20.0. Descriptive statistics and binary logistic regression were used for the analysis. Adjusted odds ratio with its 95%CI was used to present strength association. A P -value of <0.05 was used to determine statistical significance of the association. Results Age <15 years (AOR=3.72, 95%CI: 1.70–8.12), baseline CD4 count <100/mm 3 (AOR=2.31, 95%CI: 1.05–5.08), TDF-3TC-NVP regimen (AOR=2.81, 95%CI: 1.28–6.18), nondisclosure of HIV status (AOR=6.78, 95%CI: 3.24–14.15) and history of poor adherence (AOR=11.19, 95%CI: 5.65–22.15) were factors independently associated with virological failure among the study participants. Conclusion Younger age, low baseline CD4 count, TDF/NVP containing regimen, poor adherence, and nondisclosure of HIV status were significant determinants of virological failure. Therefore, adherence counseling and paying due attention to patients having these determinants are required to achieve adequate viral load suppression.