An investigation was determined the effectiveness of using Arbuscular Mycorrhizal Fungi (AMF) to enhance plant growth and protect host plants from fungal pathogen diseases. The study had also identified the best substrates for producing AMF spores for biofertilizer products. First, the dominant AMF species were isolated from soils where corn, pepper, and coffee were grown. They were identified as Acaulospora longula (Aca1) and Gigaspora marganita (Gig2, Gig3). These AMF spores were added to corn seeds, and after three days, fungal pathogens were introduced. The results showed that all AMF species promoted corn seed germination and plant growth. The best substrate for Aca1 to amplify its spores in host plant was MT5, consisting of soil, sand, and coconut fiber in the ratio of 1:1:1. Meanwhile, MT2, consisting of 2 parts soil, 1 part sand, and 1 part coconut fiber, was found to be the best substrate for Gig2 and Gig3; numbers of AMF spores were 487.5, 351.5, and 350.00 spores per 100 g substrate, respectively. These AMF spores’s production were then tested for their effect on Phytophthora capsici Leonian, Fusarium oxysporum Schltdl, and Rhizoctonia solani Kuhn, causing root rot disease on Piper longum L., Coffea arabica L., and smut disease in Zea mays L. The results showed that AMF could protect plants from fungal pathogen diseases, reducing the number of infected host plants by 35 % after 3 months of inoculation in P. longum, 10 % in C. arabica, and 33.4 % in Z. mays.