To test the effect of substrate acidolysis on litter decomposition of Cunninghamia lanceolata needles and Cinnamomum camphora leaves, we conducted a field experiment in western Hu'nan Province, China. In general, the results showed that mass loss of litter was stimulated by substrate acidolysis, and increased with the magnitude of acidolysis. Moreover, the effect of acidolysis on C. camphora litter was stronger than that on C. lanceolata litter. We found that acidolysis may affect the degradation of C. lanceolata litter through inhibition of microbial activities, including fungal biomass and cellulolytic and ligninolytic enzymes. However, for C. camphora litter, the responses of microbial activities to acidolysis during decomposition depended on the measured variables and decomposition stages. The responses of total mass loss, lignin loss, and cellulose loss of degrading litter to acidolysis differed between the two species, and depended upon decomposition stage (i.e., early and late stages of litter decomposition). Regarding the litter of C. lanceolata, the mass loss declined with the strengthening of substrate acidolysis (T1 > T2 > T3) at the early stage of decomposition, whereas it improved with increasing acidolysis (T3 > T2/T1) at the late stage of decomposition. For the litter of C. camphora, the opposite was the case at each stage of the decomposition process. Thus, litter substrate acidolysis not only altered organic chemical components and physical compactness, but also altered the microbial community during the processes of litter decomposition. These combined effects, at different levels, determined the responses of litter decomposition and associated microbial activities to substrate acidolysis of litter.