ABSTRACT The soil microbial communities involved in the biogeochemical cycles of plant nutrients are negatively affected by unfavorable agricultural practices. In three tillage systems (traditional tillage, TT; traditional tillage with residue incorporation, TTI; and conservation tillage, CT) with three crop rotations (cereal-cereal, C-C; legume-cereal, L-C; and cereal-legume, C-L) at three soil depths (0-5, 5-15, and 15-30 cm), the effects on the populations of bacteria, actinomycetes, and fungi over a period of six crop cycles (3 yr) were evaluated. The tillage system, crop rotation, and depth affected the concentration of microbes in the soil. Under TT/C-C (regional control), they decreased by 7.5%; in contrast, under CT/L-C and TTI/L-C, they increased by 144% and 76%, respectively. Regardless of the tillage system, rotation with legumes, especially when the legume was cultivated in the spring-summer cycle (C-L), caused significant increases in microbial populations. At the end of 3 yr, under CT and TTI the populations of actinomycetes increased, while the fungal population remained stable and the bacterial populations fluctuated in the different crop cycles. In all treatments, the concentration of microorganisms decreased with soil depth. Local practices represent a risk to the diversity of soil microbiota, and it is imperative that farmers adopt conservation practices to achieve sustainability.