Soil microbial communities are key indicators of changes in soil quality caused by cropping systems. Although there are studies that prove that coffee agroforestry systems increase or maintain soil organic matter levels, the effect of microbial biomass and enzymatic activity in tropical regions, especially in places with high altitude and mild temperatures, is still poorly understood. Thus, the present study aimed to evaluate the influence of two agroforestry systems (AFS-G: coffee intercropped with Grevillea robusta, AFS-C: coffee intercropped with Toona ciliata) and Coffea arabica monoculture on soil microbial biomass and activity and on the arylsulfatase and β-glucosidase enzyme activities, taking a native forest (Semi-deciduous tropical forest, with a predominance of the genera Parapiptadenia and Anadenanthera) as the control system. This study was performed in Oxisol soil areas located in the municipality of Barra do Choça, Bahia, Brazil. Litter (L, F and H layers) and soil topsoil (0–10 cm depth) samples were collected. Both were collected with four repetitions (four composite samples) in each system, litter with a 0.0625 m2 template and soil using a Dutch auger. The carbon microbial biomass, microbial activity (CO₂ released), arylsulfatase and β-glucosidase enzyme activities, organic carbon (SOC) and total nitrogen were also analyzed in the soil samples. The coffee with Grevillea robusta agroforestry system (AFS-G) did not reduce SOC compared to native forest, but the coffee with cedar agroforestry system had SOC losses at a rate of 0.56 Mg ha−1 year−1. The AFS-C cultivation system presented more MB-C (468.43 µg g−1) when compared to the other coffee systems. The AFS-G maintained the microbial activity levels and β-glucosidase and arylsulfatase enzyme activities in relation to the native forest. Coffee agroforestry systems (both AFS-G and AFS-C) in a tropical high-altitude climate region in Brazil are more favorable to maintain microorganisms and their activity in the soil, and therefore should be encouraged in order to more sustainably manage coffee growing.