Background. HIV testing is an important step for entry and linkage into HIV care. Utilization of HIV testing services among transport workers may be challenging, because of the mobile nature of their jobs. We assessed utilization of HIV testing services and identified factors influencing the utilization of HIV testing services among motorcycle taxi (boda-boda) riders in Fort Portal Municipality, Kabarole District, Southwestern Uganda. Methods. We conducted a cross-sectional study among boda-boda riders, aged 18 years and above, from July 15 to July 29, 2020. We recruited participants through simple random sampling. Data were captured using a self-administered questionnaire. Binary logistic regression was used to identify factors associated with utilization of HIV testing services. Results. Of the 315 participants who received questionnaires, 305 (97%) responded. The mean age of the participants was 32 (±7.1) years and ranged from 18 to 55 years. Of the 305 participants, 238 reported having taken an HIV test and received results in the past 12 months, for an HIV testing utilization rate of 78.0% (95% CI: 73.0–82.6%). In multivariable analysis, participants who were less likely to utilize HIV testing services were those aged ≥30 years ( aOR = 0.33 ; 95% CI: 0.16–0.70, P = 0.004 ) and those who had HIV-related stigma ( aOR = 0.27 ; 95% CI: 0.08–0.88, P = 0.030 ). Participants who were more likely to utilize HIV testing services were those who knew HIV status of their primary partners ( aOR = 4.23 ; 95% CI: 1.24–14.49, P = 0.022 ) and those who had good knowledge on HIV/AIDS ( aOR = 3.94 ; 95% CI: 1.65–9.41, P = 0.002 ). Conclusions. Utilization of HIV testing services among the boda-boda riders in Fort Portal Municipality, Southwestern Uganda, was high. More efforts should focus on targeting older boda-boda riders for HIV testing, reduction of HIV-related stigma, improving knowledge on HIV/AIDS, and encouraging communication and disclosure between partners, in order to consolidate the gains made in HIV testing services in this bridge population.