Chloroform is commonly used as an organic solvent to extract phospholipid fatty acids from soil samples. However, the extraction efficiency of the fatty acids depends on the particular chloroform stabilizers used. The effect of chloroform stabilizers 2-methyl-2-butene and ethanol was investigated at different steps of the extraction procedure. Only the ethanol stabilized chloroform prevented the loss of certain phospholipid fatty acids. In particular, the unsaturated fatty acids 16:1ω7c, 16:1ω6c, 16:1ω5, 17:1ω8, 18:1ω7c, 18:1ω5, the fungal biomarker 18:2ω6,9 and the saturated fatty acid 17:0 were absent when chloroform stabilized with 2-methyl-2-butene was used. In addition, the total phospholipid fatty acid concentrations were also significantly reduced when chloroform stabilized with 2-methyl-2-butene was used. Thus, the proper choice of chloroform stabilizer for the analysis of phospholipid fatty acids is very important.