Mycorrhizal association increases the absorption of water and nutrients by plants, and thus, affects their metabolism. This study aimed at assessing the single and conjoint effects of the arbuscular mycorrhizal fungus (AMF) Claroideoglomus etunicatum and soil’s humic substances (HS) on the composition of essential oil (EO), its antimicrobial activity, and development of basil (Ocimum basilicum L.). The experimental design was completely randomized in a 2 x 2-factorial design (with and without AMF and HS) with 10 replications as follows: T1 - without AMF inoculation and without HS addition; T2 - with AMF inoculation and with HS (2 L/ha); T3 - without AMF inoculation and with HS; T4 - with AMF inoculation and with HS. Plants were grown in pots with 5 kg sterile soil in a greenhouse for 6 months. Then, they were harvested and evaluated for spore density, AMF root colonization, shoot dry matter (SDM), yield and chemical composition of EO as well as minimum inhibitory concentration, used to compare the antimicrobial effect of EO in nine bacteria. The addition of HS to the soil increased the spore density of AMF and SDM (P < 0.001). The AMF inoculation with and without HS increased the EO yield of basil (P < 0.001). The treatments altered the composition of the basil EO, in which a total of fourteen compounds were detected. In treatments with AMF and/or HS, linalool (28.64 to 42.94%) and estragole (27.53 to 47.17%) were the major compounds. However, in the control (without AMF and HS), eugenol (33.90%) was the major compound. The EO showed low microbial activity against bacteria Bacillus cereus, Salmonella enterica serovar typhi, Shigella flexneri, Klebsiella pneumoniae, and Xanthomonas axonopodis pathovar begoniae. AMF inoculation and HS addition could improve the development of basil, increase EO yield, alter the EO chemical composition and its major compounds, but presented low or none microbial activity against most of the bacteria tested in this study.