Traditional healers in Thailand are a primary source of health care for the Thai people. Highly experienced traditional healers are generally older people and they continue to pass away without recording or passing on their knowledge. Consequently, the cumulative knowledge held by traditional healers regarding the use of medicinal plants is being eroded and could be lost. In this study, we aimed to identify and document the medicinal plants and associated ethnobotanical knowledge held by traditional healers in Roi Et in northeastern Thailand. Data and plant specimens were collected from four traditional healers of the Phu Tai people. They were selected by purposive sampling and questioned using a semi-structured interview. The interviews covered their training, the ailments treated, treatment techniques, method of preparation and in addition, several healing sessions were observed. During field walks, we searched for the medicinal plants with the healers to review and document the availability of medicinal plants at each locality and in different habitats around the villages. Use values (UV) were calculated to estimate the importance of each medicinal plant and informant agreement ratios (IAR) were calculated to understand how widely known the uses were. The four Phu Tai traditional healers knew 162 medicinal plant species in 141 genera and 63 families. The family with the most medicinal plants was Leguminosae with 15 species. The plant part that they used most commonly was the stem, which was used for 82 species (49%). The most common preparation method was decoction, which was done for 124 species (75%). The most important and widely used medicinal plants were Rothmannia wittii, which had the highest use value (UV = 1.7). Most medicinal plants were used for treating tonic (34 species (21%)). Jaundice had the highest informant agreement ratio (IAR = 0.5). The most common life form among the medicinal plants was trees (56 species (34%)). The medicinal plants were mostly collected in community forests (81 species (49%)). Considering the richness of the healer’s pharmacopeia, and the fact that their profession is not being perpetuated, this study points to the urgent need to document the traditional knowledge from the old herbalists before it disappears with the last practitioners from rural communities in Thailand.