The common practice of maize monoculture combined with non-retention of crop residue limits the soil health status of the African smallholder's no-till systems. Therefore, the study evaluated crop rotation and residue management effects on soil health indicators: particulate organic matter (POM), soil organic carbon (SOC), microbial biomass carbon (MBC) and activities of fluorescein diacetate, beta-glucosidase, arylamidase and acid phosphates under no-till at Phandulwazi and University of Fort Hare (UFH) representing sub-humid and semi-arid, respectively. The crop rotations; maize (Zea mays L.)-fallow-maize (MFM), maize-fallow-soybean (Glycine max L.) (MFS); maize-wheat (Triticum aestivum L.)-maize (MWM) and maize-wheat-soybean (MWS) and residue managements; removal (R-) and retention (R+) were evaluated. The UFH consistently had higher mean values of the soil health indicators across the treatments compared to Phandulwazi. Contrast analysis showed significant (P < 0.05) improvement of POM, SOC, MBC and enzyme activities with both residue retention and legume rotation compared to residue removal and cereal-only rotations. The SOC from the various crop rotations was in the order: MWS > MFS > MWM > MFM across the sites. The improvements in soil biological parameters indicated a potential for soil health and soil quality improvement with the adoption of residue retention and legume rotations in the maize-based no-till systems in the semi-arid and sub-humid agro-ecologies of the Eastern Cape, South Africa.