Arbuscular mycorrhizal fungi (AMF) can be used as microbial fertilizers. Hydroponic cultivation offers significant advantages by reducing factors that cause damage during traditional soil-based cultivation. Furthermore, mycorrhiza helper bacteria (MHB) can improve mycorrhizal fungal colonization. The objective of this study was to examine the function of Funneliformis mosseae in promoting rice growth under soilless culture using a hydroponic cultivation system with two different levels of nutrient supply: A and B (high and low levels). Furthermore, Bacillus methylotrophicus was isolated from Funneliformis mosseae spores to examine its potential to promote rice growth in combination with AMF. The findings illustrated that three-month-old rice plants at the ripening stage inoculated with Funneliformis mosseae showed the highest increases in height, fresh weight of leaves and roots, and dry root weight under low levels of nutrient supply, with successful AMF root colonization of 78.67%. Additionally, co-cultivation of Funneliformis mosseae and Bacillus methylotrophicus significantly enhanced plant height, fresh weight of leaves and roots, and dry root weight under low levels of nutrient supply compared to uninoculated plants under high levels of nutrient supply. High levels of nutrient supply significantly decreased Funneliformis mosseae colonization of rice roots. These findings suggest that optimal levels of nutrient supply are necessary for rice growth with AMF colonization because high nutrient supply restricts plant growth and reduces AMF root colonization. However, Bacillus methylotrophicus did not demonstrate a strong relationship with AMF colonization. Based on these findings, AMF can be applied to hydroponic cultivation systems. Nevertheless, the MHB-AMF relationship should be studied further.