Aim: The present study aimed to document the traditional knowledge of medicinal plants and antimicrobial validation of Thespesia populnea used by Yanadi tribe of Chittoor District, Andhra Pradesh, India. Materials and Methods: The study was mainly focused on documentation of medicinal plants used by Yanadi tribe to treat different diseases with a standard questionnaire. These plants were cross checked in Dr. Dukes database and available literature to know the significance of this tribe on medicinal knowledge. Among the documented plants T. populnea was selected for antimicrobial activity with disc diffusion assay. Results: Among the documented medicinal plants, herbs were the most utilized plants, followed by leaf part of the plants; paste form of medicinal preparation was the dominant one among the mode of preparations and oral administration was generally followed by this tribe. When checked these plants in Dr. Duke’s phytochemical and ethnobotanical database most of the medicinal plants were matched at least one medicinal use and most of them were correlated with existing literature. In antimicrobial activity, the microbial pathogens K. pneumonia among bacteria and R. arrhizus among fungi were most susceptible to methanol extract of T. populnea. Conclusion: From this study, we conclude that the preparation and dosage of the medicines by Yanadi tribe of this area is unique and the correlation of medicinal data with Duke’s database and existing literature reveals high medicinal significance of claimed data of this tribe and potential inhibitory activity of T. populnea could be studied further to isolate effective antimicrobial agents. [J Complement Med Res 2017; 6(2.000): 158-169]