There is growing recognition for the need to study the impact of agricultural land uses on biological and biochemical properties of soils. In Madagascar, cropping systems based on direct seeding with permanent vegetation cover provide a new means for sustainable agriculture to protect the environment and make the most of natural resources. This study assessed the effects of different direct seeding mulch-based cropping systems on soil microbial biomass and activities. The soil was andic Dystrustept. Samples of the soil were taken from 0 to 5 cm soil layer of three direct seeding mulch-based cropping systems (DMC using crop residues and living mulches). The samples were compared with samples from conventionally tilled plots (CT) and natural fallows (NF). The field experiments were carried out over a 12-year-period and two types of amendment were applied once a year at sowing, farmyard manure (FYM) and farmyard manure combined with an NPK chemical fertilizer. The C and N content, microbial basal respiration and biomass and β-glucosidase, urease and acid phosphatase activities were determined. The results showed that there was no interaction between soil management strategies and the use of fertilizer. Furthermore, the fertilizer did not affect the soil C and N content or the acid phosphatase and urease activities. Farmyard manure with added NPK had a significantly greater effect than farmyard manure on its own, increasing the microbial biomass, soil respiration and β-glucosidase activity up to 26%, 52% and 20%, respectively but there was no significant difference between natural fallows and direct seeding mulch-based cropping systems. However, conventional tillage showed a significantly lower soil microbial biomass, C content, microbial respiration and urease activity than natural fallows. The results for direct seeding mulch-based systems varied according to the microbial activities measured. However, soil β-glucosidase and acid phosphatase activities were significantly higher for the direct seeding mulch-based systems using crop residues than for the direct seeding mulch-based systems using living mulches. Direct seeding mulch-based systems with Desmodium uncinatum living mulch had significantly lower microbial biomass and respiration than the other direct seeding mulch-based systems. There was less evidence of change in the soil between natural fallow and direct seeding mulch-based systems but a higher build-up of some microbial properties was obtained for direct seeding mulch-based systems soil than in conventionally tilled soils and natural fallow.