People living with HIV often experience mental health disorders and engage in substance use. Evidence, however, is limited about the influence of mental health disorders and substance use on non-adherence to ART. We conducted a cross-sectional study among 682 HIV-positive people on ART in Nepal. We measured their depressive symptoms, anxiety, stress levels, substance use, and non-adherence to ART. We developed logistic regression models to examine the association of mental health disorders and substance use with non-adherence to ART. Experiencing depressive symptoms was positively associated with ART non-adherence among HIV-positive people (men: AOR = 2.77,  = .001; women: AOR = 3.69,  = .001). Additionally, both men and women were more likely to have non-adherence to ART when they had anxiety (men: AOR = 2.19,  = .022; women AOR = 2.83,  = .001) and higher stress scores (men: AOR = 1.11,  = .001; women: AOR = 1.08,  = .001). While substance use was associated with non-adherence only in HIV-positive men (AOR = 3.12,  < .001). Depressive symptoms, anxiety, and high level of stress had negative roles on adherence in HIV-positive men and women. While substance use had a negative role on ART adherence among men only. Results highlight that the HIV-positive people should be screened and provided treatment and psychosocial support while providing ART services to improve their medication adherence.