The study characterizes and compares the structural and functional dynamics of mite communities in sugarcane fields in Ayala, Morelos, Mexico, and evaluates their potential as soil-quality bioindicators through analyses of vertical and temporal stratification. Soil samples were collected from four plots (three under active sugarcane cultivation, one non-cultivated) on four dates in 2025 (March, June, Sept., Dec.). At each plot, soil cores were taken at two depth intervals (0–5 cm and 5–10 cm) and abiotic variables recorded. Mites and springtails were extracted with Berlese–Tullgren funnels; microarthropods were separated, counted, mounted, and identified to family level. We calculated abundance, richness, alpha and beta diversity, and vertical stratification patterns, and compared these metrics across land- use types and depths. From 224 samples we recorded 12,191 specimens, of which Acari dominated (n = 8,512; 69.8%). Oribatida were the most abundant order (20.5%), followed by Prostigmata (17.5%), Mesostigmata (11.9%), Endeostigmata (9.2%), and Astigmatina (2.1%). Mites belonged to 39 families in total: 20 Oribatida families, 6 Mesostigmata, 11 Trombidiformes (Prostigmata), and 2 Endeostigmata. Acari abundance was markedly higher in the 0–5 cm layer (6,109 individuals) than in the 5–10 cm layer (2,403 individuals), indicating a strong surface-layer dominance. However, family richness was greater in the deeper horizon (5–10 cm). Fisher’s alpha was higher at 5–10 cm (a = 1.032) than at 0–5 cm (a = 0.657), pointing to greater relative diversity at depth. Community composition was largely homogeneous among agricultural plots: plots 2 and 3 showed the highest similarity (Jaccard J = 0.844). The non-cultivated plot exhibited the lowest similarity to cultivated plots; the largest compositional turnover was between plot 2 and the non-cultivated plot (turnover = 0.444), indicating pronounced taxonomic differences associated with land use. Overall, agricultural management significantly alters mite community structure, supporting the use of mites as sensitive bioindicators of soil quality in sugarcane agroecosystems.