The discussion on medicinal plants is becoming more relevant these days, specifically within the realm of exploring secondary metabolites with phytopharmaceutical properties to fight against the alarming crisis of antibiotic resistance. In conjunction with the host plant, endophytes are recognized for their ability to produce antimicrobial compounds and potentially trigger an increase in the production of the host plant secondary metabolites. For that reason, it is essential to investigate the structure and composition of the endophytes community of medicinal plants to unravel more about their potential related to the production of antimicrobial compounds. In this chapter, we delve into a step-by-step protocol to gain insight into the endophytic population of medicinal plants, utilizing the powerful and rapid approaches of next-generation sequencing (NGS) technology. The processing of plant material is described and followed with appropriate methods for nucleic acid extraction, amplification of the marker gene, purification, sequencing, and data analysis. The knowledge from the data might be utilized further to design a suitable approach in the finding of novel antimicrobial compounds, addressing the global challenge of antimicrobial resistance.