Soil microbial community and soil physiological parameters were investigated by analyzing phospholipid fatty acids extracted from the soils amended with chitosan powder and solution in a cucumber greenhouse. The soils were sampled at 90, 160, 200 days after treatment. Identified fatty acids were analyzed with principal component (PC) analysis. Chitosan powder soils and chitosan solution soils were separated from non-treated control soils by PC1 and PC2 90 days after treatment, respectively. And chitosan powder soils were separated from non-treated control soils by PC2 160 days after treatment. The ratio of fungi to bacteria increased significantly in chitosan solution-amended soils compared with the control soils 90 days after treatment. Microbial groups and physiological parameters were investigated 160 days after treatment: vesicular-arbuscular mycorrhizal fungi (VAM) significantly increased in soils amended with chitosan powder compared with other soils, the ratio of gram negative bacteria to gram positive bacteria and cyclo-fatty acids to precursors were significantly higher and lower in soils amended with chitosan solution and chitosan powder compared with control soils, respectively, and the ratio of fungi to bacteria were significantly lower in control soils compared with chitosan-treated soils. The chitosan powder increased the ratio of aerobic to anaerobic bacteria and lowered the ratio of saturated to unsaturated fatty acids compared with chitosan solution 200 days after soil application. In conclusion, chitosan powder changed the soil microbial community and the effects maintained up to 160 days after soil application. The effect of physiological parameters on the soil microbial community started to appear 160 days after and continued up to 200 days after soil application of chitosan.