Aims: A field experiment was conducted to evaluate the effects of alley cropping systems on microbial activity and soil organic matter (SOM) pools. We hypothesized that enzyme activity and labile pools of SOM are early and sensitive indicators of changes induced by tree introduction in the cropping systems. Methods: Poplar-alfalfa and alder-gramineous (cereal or ryegrass) associations and their respective control systems (alfalfa and gramineous) were compared in terms of soil carbon (C), nitrogen (N) and water contents, SOM labile pools, NIRS-MIRS spectra and microbial enzyme activity in the topsoil (0–15 cm) for 4 years after tree planting. Results: After 1 year, tree introduction induced a decrease in soil water content, microbial biomass N and some enzyme activities under alfalfa system. After 4 years, tree introduction resulted in higher soil water contents in both systems (alfalfa and gramineous); higher microbial biomass N and lower C:N in alfalfa-poplar plots compared to control plots. MIRS-NIRS analyses showed a greatest differentiation in SOM quality between alfalfa-based systems. Conclusions: The effects of temperate agroforestry systems on SOC in the topsoil are relatively weak in the first years after tree introduction. Observed effects were more pronounced in the alfalfa-poplar system, probably due to higher tree growth. Further studies will provide insights into the longer-term effects of these systems on soil functioning.