Refining the Study of Endophytic Fungi: Best Practices in Antibacterial Research.

As multidrug-resistant bacteria continue to emerge rapidly, the demand for bioactive molecules with alternative mechanisms of action become increasingly urgent to address this global health crisis. Endophytic fungi (EFs) have emerged as promising sources of antimicrobial metabolites due to their remarkable biosynthetic potential. However, research on EF-derived antimicrobials often faces significant methodological limitations, including inadequate strain characterization, suboptimal bioassay design, limited quantitative validation, and insufficient chemical characterization of active compounds. These shortcomings hinder the translation of laboratory findings into clinically relevant solutions. This review provides a critical assessment of the current state of antimicrobial research involving EFs, highlights persistent methodological gaps, and advocates for the adoption of integrative and standardized approaches. In contrast to previous reviews, this work introduces a methodological framework that prioritizes reproducibility, cross-comparison of antimicrobial efficacy against bacterial targets, and the alignment of bioassay practices with clinical relevance. We argue that enhancing methodological rigor in endophytic fungal research is crucial to fully realizing their potential as a sustainable source of novel antimicrobial agents.