The present study evaluated the changes of soil microbial communities that were subjected to no-till and compared the results to those subject to tillage for organic farming in a controlled horticultural field by fatty acid methyl ester. Fungi (P < 0.001), gram-positive bacteria (P < 0.001), arbuscular mycorrhizal fungi (P < 0.01), and actinomycetes (P < 0.01) in the no-till soils were significantly larger than those in the tillage soils. The no-till in the subsoil had a significantly lower ratio of cy17:0 to 16:1ω7c compared to that of tillage, indicating that microbial stress decreased because the soils were not disturbed (P < 0.05). Fungi should be considered as a potential factor responsible for the obvious microbial community differentiation that was observed between the no-till and tillage areas in a controlled horticultural field.