Ester-linked fatty acid methyl ester (EL-FAME) profiles were used to describe differences in soil microbial communities influenced by conventional farming system (CFS), and organic farming system (OFS) in controlled horticultural land. Soil physicochemical properties and soil microbial communities were determined in the experimental fields. Higher organic matter content in OFS reduced soil bulk density which in turn increased the soil porosity. Generally, soil chemical properties in OFS were higher than those of CFS, but EC value in OFS was significantly lower than that of CFS. With the exception of Fe content, other macronutrient contents and pH in both farming system decreased with the soil depth. Soil microbial biomass of OFS was approximately 1.3 times in topsoil and 1.8 times in subsoil higher than those of CFS. Lower ratios of cy17:0 to $16:1{omega}7c$ and cy19:0 to $18:1{omega}7c$ were found in the CFS soils than the OFS soils, indicating that microbial stress decreased. The ratio of MUFA to SFA was higher in OFS due to organic input to the soil. In principal components analysis (PCA), the first variable accounted for 54.3%, while the second for 27.3%, respectively. The PC1 of the PCA separated the samples from CFS and OFS, while the PC2 of the PCA separated the samples from topsoil and subsoil. EL-FAMEs with the positive eigenvector coefficients for PC1 were cy17: 0 to $16:1{omega}7c$ ratio, cy19:0 to $18:1{omega}7c$ ratio, soil pH, soil organic matter, and soil $NO_3$-N content. Our findings suggest that the shifting cy19:0 to $18:1{omega}7c$ ratio should be considered as potential factors responsible for the clear microbial community differentiation observed between different cultivation systems and soil depth in controlled horticultural land.