Objective We evaluated diagnostic performance of oral swab analysis (OSA) for tuberculosis (TB) in a high HIV/TB burden setting in Kenya. Methods In this cross-sectional study, buccal swabs and sputum were collected from 100 participants with suspected TB in outpatient clinics in Kenya at enrollment and subsequent morning visits. Buccal swabs underwent IS6110-targeted qPCR analysis. Sputum was evaluated by Xpert MTB/RIF (Xpert) and culture. Diagnostic performance of OSA for TB diagnosis was evaluated relative to a combined reference of sputum Xpert and culture. Results Among 100 participants, 54% were living with HIV (PLHIV). Twenty percent (20/100) of participants had confirmed TB (19/20 [95%] culture-positive, 17/20 [85%] Xpert-positive). Overall buccal swab sensitivity was 65.0% (95% CI 40.8–84.6%) vs. sputum Xpert/culture and 76.5% (95% CI 50.1–93.2%) vs. sputum Xpert alone. Specificity was 81.3% (95% CI 71.0–89.1%) and 81.9% (95% CI 72.0–89.5%) compared to sputum Xpert/culture and Xpert alone, respectively. Sensitivity among PLHIV (n = 54) with suspected TB was 83.3% (95% CI 35.9–99.6%) vs. sputum Xpert/culture and 100% (95% CI 47.8–100.0%) vs. sputum Xpert alone. Among participants with TB, mean OSA threshold quantitation cycle (Cq) value was lower (stronger signal) at subsequent morning compared to enrolment visit (33.4 SD ± 3.7 vs. 35.2 SD ± 2.9, p = 0.009). Conclusions In this pilot study, results confirm M. tuberculosis DNA is detectable in oral swabs including among PLHIV with fair diagnostic performance. Further work is needed to optimize OSA and evaluate its utility in diverse settings.