Sugarcane crop conventional tillage has been replaced by deep soil preparation with few studies about its effect on soil quality and sustainability. The aim of this study was to assess structural volumes ina dystrophic Red-Yellow Latosol subjected to conventional tillage (CT) and deep localized soil tillage (DLST) to verify how the microbiological parameters were affected. The study was conducted in a soil derived from the Caiuá Sandstone formation in Brazil. Four trenches were dug for each soil tillage system to describe the cultural profile and evaluate carbon microbial biomass (CMB) and nitrogen (NMB), basal respiration (BR), and metabolic quotient (qCO2). CT profiles exhibited a predominance of cracked soil volumes, medium-sized and large compact clods with some porosity, and continuous volumes with no cracks and of intermediate porosity. DLST profiles were predominantly free-soil volume with no cohesion and porous in appearance, and compact, cohesive volumes with no porosity visible. The highest levels of CMB and NMB were in the cracked soil under CT. Higher microbial activity indicated by BR and qCO2 were in the free powdery soil under DLST. Soil pulverization caused by DLST could cause serious consequences on soil functionality, boosting erosion, and metabolic stress in the microbiota.