Coffee seedlings are commonly produced on substrate composed of a mixture of soil and cattle manure, supplemented with chemical fertilizers. Alternatives to reduce production costs and produce seedlings of greater quality and health include the use of commercial organic substrates, which require less handling. The use of beneficial microorganisms such as arbuscular mycorrhizal fungi (AMF) can be considered a good alternative for production of more vigorous coffee seedlings. The main goal of this study was to evaluate the effect of the inoculation of AMF isolates on coffee seedlings development in a commercial organic substrate (based on coconut fiber) and conventional substrate (mixture composed of soil and cattle manure compost). Ten AMF were tested: Rhizophagus irregularis, Glomus macrocarpum, Claroideoglomus etunicatum, Rhizophagus clarus, Glomus spp., Gigaspora margarita, Acaulospora morrowiae, Acaulospora scrobiculata, Acaulospora spp., and Dentiscutata heterogamma. Plant growth, shoot P content, mycorrhizal colonization, extraradical mycelium length, phosphatase activity, and photosynthetic pigments were evaluated. The effects of mycorrhization depended on both the inoculated fungal species and the substrate for seedling cultivation. Inoculation of G. margarita, Acaulospora spp., and Glomus spp.in the conventional substrate conferred the best growth plant responses, increasing shoot biomass by 160 to 320%. In the commercial substrate, the most efficient AMF were R. clarus, Glomus spp, A. morrowiae and A. scrobiculata, with up to 149% of shoot biomass increase.The commercial organic substrate and the inoculation of some of the AMF isolates were highly beneficial to coffee seedlings development and can replace the use of the conventional substrate. These results open new opportunities for the use of AMF as an inoculant to improve coffee seedling production in commercial organic substrates.