Eswatini has the highest global prevalence of HIV despite universal access to free treatment. Lack of compliance continues to be a significant challenge for HIV care and management programs throughout the country. Studies investigating barriers to antiretroviral therapy (ART) adherence, particularly in women, who are the most vulnerable to HIV infections, are limited. The disproportionate impact of HIV on women can be attributed to multiple risk factors at the individual, household, and community/structural levels. Women living in rural settings are particularly at risk as a result of socio-cultural and environmental vulnerabilities such as oppressive traditional practices, intimate partner violence, and limited access to financial resources. We employed a mixed methods exploratory sequential design to investigate barriers associated with ART adherence among HIV-infected women living in rural Eswatini. The qualitative phase of the study included four focus group discussions (FGDs n=41) with HIV-infected rural women, and in-depth interviews of healthcare workers (n=8). Data analysis used open and axial coding techniques, with interpretation following the social ecological model. The quantitative phase involved in-person survey interviews of HIV-infected women (n=166) and logistic regression models were used to examine the extent to which critical barriers affected ART adherence. Intrapersonal barriers to ART adherence include: hunger, side effects of ART, personal stress, stigma, and forgetfulness. Lack of food, lack of disclosure of HIV status, and limited and financial resources were identified as critical barriers at the household level. Community/ institutional barriers included: lack of privacy, travel time, transportation costs, excessive alcohol use, maltreatment, gossip, and long waits at clinics. Of twenty barriers identified from the qualitative phase, nine (hunger, hunger-related medication side effects, feelings of stress, forgetfulness, mode of transport, age, gossip, maltreatment by clinic staff, and community support) were significantly associated with ART adherence. Numerous factors across multiple levels interact and influence ART adherence. Collectively, our findings pinpoint priority areas to target for development, testing, and evaluation of future interventions aimed at increasing HIV medication adherence among one of the most vulnerable segments of Eswatini’s population, HIV-infected women in rural communities.