As a biocide of soil fauna, naphthalene has been widely used in the study of the ecological functions of soil fauna; however, their effect on non-target organisms has raised doubts about its widespread application. The present study was conducted in order to determine whether there are non-target effects of naphthalene in the soil of subalpine forest in the west Qinghai-Tibet Plateau. The short-term responses of soil microbial biomass, microbial abundance, and microbial phospholipid fatty acids (PLFAs) to naphthalene were studied in microcosms. The results showed that the soil microbial biomass carbon (MBC), fungal abundance, and the contents of bacteria, fungi, gram-positive bacteria (G⁺), and gram-negative bacteria (G^-) PLFAs were reduced during the entire study period. The lowest MBC and G⁺ PLFAs contents in both the no-naphthalene and naphthalene microcosms were observed at the end of culture (52 days), and the lowest bacterial, fungal, and G^- PLFAs contents in both microcosms were observed at 45 days after application of naphthalene. The content of microbial biomass nitrogen (MBN) in the naphthalene and no-naphthalene microcosms showed increased and decreased dynamics during the entire study period, and the highest MBN in both microcosms were observed at 17 days after application of naphthalene, but reverse dynamic changes were observed for the ratio of MBC/MBN. The ratio of fungi/bacterial PLFAs in the no-naphthalene microcosm showed decreased and increased dynamics during the entire study period, and the highest fungi/bacterial PLFAs were observed at 17 days after application of naphthalene, whereas little change was detected in the naphthalene microcosm. The ratio of G⁺/G^-PLFAs in the naphthalene microcosm was reduced during the entire study period, but showed increased and decreased dynamics in the no-naphthalene microcosm during the entire study period. Naphthalene treatment only significantly affected the ratio of G⁺/G^-PLFAs, whereas the interaction of naphthalene treatment and sampling time had significant effects on the MBC/MBN ratio, bacterial abundance, fungi/bacterial abundance, PLFA content of bacteria and fungi, ratio of fungi/bacterial PLFAs, and G⁺/G^- PLFAs ratio. In the short term, the non-target effects of naphthalene as a biocide to reduce soil fauna abundance may have a temporally variable influence on soil microbial communities in the subalpine forests of western Sichuan.