Background In Nigeria, similar to most of sub-Saharan Africa, the fight against HIV/AIDS is hampered by various barriers. Particularly, key populations (KP) face unique challenges in accessing antiretroviral therapy (ART), attributed to health systems, patient-related, and community-related factors. This study aims to explore these barriers among KP in Nigeria, enhancing understanding to improve ART access and outcomes, thereby contributing to global efforts to end the HIV/AIDS epidemic. Objectives The objective of this study is to explore barriers to accessing ART services among KP living with HIV in the southwest region of Nigeria. Materials and methods This descriptive cross-sectional study, which was carried out in the southwestern Nigerian states of Ondo and Ogun, involved 4,253 KP attending ART clinics. Using a multistage sampling technique, 219 females and 154 males between the ages of 17 and 72 were chosen. Semi-structured survey questions were used to get information to elicit these barriers. SPSS Statistics (version 29.0; IBM Corp., Armonk, NY) was used for quantitative data analysis. Results Specifically, 48.3% of respondents were men, and 58.7% were women; 54.0% were female sexual workers, and the next highest percentages were men who have sex with men (27.0%) and injectable drug users (19.3%); and 79% (51.0% agreed, 28.0% strongly agreed) concurred that the barriers to accessing ART are transportation, distance, and financial challenges. Patients at ART clinics were generally satisfied with the healthcare and health workers' attitude. Measures to protect privacy and confidentiality were considered crucial by the respondents. There are significant associations between ART accessibility and socio-demographic and socio-economic characteristics (p-value <0.05). Conclusion This study probed the complex landscape of barriers to ART access faced by KP in Nigeria, underscoring the critical need for tailored, innovative strategies to overcome these obstacles and offering actionable insights for stakeholders to enhance ART adherence and access thereby significantly improving the quality of life for people living with HIV.