AbstractPlant roots are associated with numerous and diverse types of beneficial and pathogenic microorganisms. Plant growth–promoting (rhizo)bacteria (PGPB or PGPR) are isolated from plants crops worldwide, and many of them are used as agricultural inoculants. Agricultural biofertilization and biocontrol of pathogens are eco‐friendly alternatives to chemical usage and have less energy, environmental, and economic costs. PGPB isolation and evaluation are essentials steps for determining bacteria that are able to improve plant development and productivity. In this unit, we present protocols to isolate bacteria from soil and plant roots (“putative” diazotrophic and endospore‐forming bacteria), as well to evaluate some of their beneficial characteristics for the promotion of plant growth (e.g., nitrogen fixation, production of indolic compounds and siderophores, phosphate solubilization, and 1‐aminocyclopropane‐1‐carboxylate deaminase activity). © 2017 by John Wiley & Sons, Inc.