The driving forces of transpiration are not only atmospheric evaporation but also root zone water supply and stomatal regulation among species. However, the biophysiological drivers of transpiration remain incompletely understood in heterogeneous karst habitats. This study investigated the commonly coexisting tree species Mallotus philippensis and Celtis biondii in two typical karst habitats: rock-dominated (RD) habitat and control soil-dominated (SD) habitat. Over 2 years, soil moisture, transpiration, root distribution, and leaf water potential were measured. The results showed that soil moisture in the RD habitat was significantly lower than in the SD habitat. Transpiration patterns also differed between habitats, with species-specific distinctions driven by biophysiological traits. M. philippensis showed small hydroscape areas and its root system mainly distributed in the soil zone in both habitats. The isohydric behaviour and lower root density in the RD habitat drove M. philippensis to reduce transpiration in response to soil water deficiency. Conversely, C. biondii had large hydroscape areas and roots capable of penetrating bedrock. It transpired higher relying on ample accessible water through anisohydric behaviour and having a more robust root system both in soil and bedrock zones in the RD habitat. Our study highlights the critical role of root water accessibility and leaf iso/anisohydric tendencies in driving transpiration.