Studies on the relationship between Handroanthus serratifolius and arbuscular mycorrhizal fungi (AMF) are limited in the literature. The influence of AMF spore density on plant development is fundamental information to determining the degree of benefits in this relationship. Therefore, the objective of this study was to investigate the effects of different AMF spore densities on thirty‐day‐old H. serratifolius seedlings, focusing on growth and biochemical parameters using a completely randomized experimental design with three different spore densities and control. The spore densities were classified as low, medium, and high, with 1.54, 3.08, and 12.35 spores g−1, respectively. Plant growth analysis, mycorrhizal colonization, nitrogen compound concentration, and carbohydrate analysis were performed. The medium spore density treatment showed the greatest increases in biomass, height, leaf area, and root volume. Furthermore, greater absorption of phosphorus and better dynamics in nitrogen metabolism were observed in mycorrhizal plants compared to the control since the ammonium and nitrate compounds were rapidly incorporated into protein and chlorophyll compounds. The carbohydrate analysis revealed the influence of source‐sink dynamics on sugar concentration in different plant parts. These findings support the importance of determining the appropriate spore density for assessing the symbiotic relationship between forest species and AMF.