Ventilation is an important measure in reducing cross-infection risk in the indoor environment. A novel ventilation concept, stratum ventilation, supplies fresh air directly to occupants’ breathing levels and creates a horizontal airflow layer, which has potential to provide high ventilation effectiveness compared with traditional air distribution system. However, the effectiveness of such system is very sensitive to the operation conditions. This study examines how occupants’ relative orientations and positions to inlet diffusers affect thermal comfort and cross-infection risks with stratum ventilation. Full-scale experiments in a test room involved life-size breathing thermal manikins representing source and target individuals. Results showed high draft risk at breathing zones was a drawback of such system. Cross-infection risks were sensitive to relative positions and orientations, with the worst scenario involving face-to-face occupants positioned in front of the inlet diffuser. The inlet air jet accelerated airborne transmission and maintained high concentrations in the close proximity.