HIV co-infection has been shown to be associated with lower tuberculosis (TB) bacterial load in studies conducted prior to widespread availability of antiretroviral therapy (ART). The purpose of this study was to determine associations between HIV co-infection and TB bacterial load, accounting for differences in time to TB diagnosis, in a high prevalence setting with widespread ART use. In Gaborone, Botswana, 186 sputum samples from people with newly diagnosed TB were tested with Xpert MTB/RIF (Xpert). TB bacterial load and time to TB diagnosis were estimated using mean Xpert cycle threshold (CT) and symptom duration, respectively. Multiple linear regression models were used to determine the associations between HIV and Xpert CT with and without controlling for symptom duration. Mean CT values were higher in people living with HIV compared to people without HIV (21.8 vs 18.6, p < 0.001). Among those living with HIV, there was a negative correlation between CD4 count and mean CT value (Spearman rho -0.27, p = 0.023). After controlling for gender, age, and symptom duration, HIV status remained an independent predictor of CT value, with an average increase of 2.0 cycles (p = 0.004) among people with HIV and CD4 count > 200 cells/mm3 and 2.8 cycles (p < 0.001) in those with a CD4 count ≤ 200 cells/mm3 compared to individuals without HIV. Increased HIV-associated immunosuppression is associated with decreased bacterial burden even in settings with widespread ART use.