ABSTRACT Phakopsora pachyrhizi causes Asian soybean rust, resulting in severe losses of soybean yield. This study assessed the biocontrol efficacy of cell-free culture filtrates and conidial suspensions of Metarhizium anisopliae (MABR-01) and M. humberi (MHBR-03) against P. pachyrhizi in vitro and in planta. A 50% concentration of culture filtrates of M. anisopliae and M. humberi inhibited the germination of P. pachyrhizi urediniospores by 85 and 96%, respectively, compared to the 50% potato-dextrose broth (PDB) control. The conidial suspensions caused no inhibition of P. pachyrhizi in vitro, but the conidial suspension of M. anisopliae controlled the rust disease in plants with a 51% efficiency. Soybean plants colonised by M. anisopliae MABR-01 and exposed to P. pachyrhizi showed less severe disease in the shoots compared to plants not colonised by M. anisopliae; the disease was reduced by 40%. Spraying plants with 50 and 75% culture filtrates of M. humberi and a 75% concentration of M. anisopliae significantly reduced rust disease, by an average of 86%. Plants pretreated with Metarhizium culture filtrates showed a 30% reduction in P. pachyrhizi colonisation based on qPCR quantification of the pathogen genomic DNA. These results indicate a high biotechnological and sustainable potential of these two Metarhizium species for control of Asian soybean rust.