To better understand the medicinal and nutritional value of mushrooms, we studied the fatty acid (FA) compositions and DPPH scavenging abilities of 11 mushrooms from Southwest China. The crude fat (CF) contents were examined initially, then 3 methods of FA methyl esterification were compared to identify which acid treatment was the most appropriate method. Then methyl esterification methods for 12 CFs were performed with acid treatment and the FA compositions were analyzed with gas chromatography-mass spectrometry. The results showed that tetradecanoic acid (14:0), hexadecenoic acid (16:1), hexadecanoic acid (16:0), heptadecanoic acid (17:0), octadecadienoic acid (18:2), octadecenoic acid (18:1), octadecanoic acid (18:0), docosanoic acid (22:0), and tetracosanoic acid (24:0) were detected in all the samples, with large amounts of hexadecanoic acid (16:0), octadecadienoic acid (18:2), octadecenoic acid (18:1), and octadecanoic acid (18:0). Daldinia eschscholtzii and Sarcodon imbricatus had the highest ratio value of unsaturated FAs to saturated FAs (4.33 and 3.03, respectively). The DPPH scavenging ability of 12 CFs was also tested. The free radical scavenging rates of the CFs were almost < 10% at a concentration of 0.10 mg/mL, except that of S. imbricatus, which reached 81.25%, with a half-maximal inhibitory concentration of 0.054 mg/mL. This strong DPPH free radical scavenging ability of S. imbricatus may be related to α-hydroxy FA.