In a diversified cropping system, the kinds of tillage methods and weed management practices significantly influence soil microbiome, which affects crop productivity. The synergetic impacts of such practices on soil microbiome in association with yield under diverse crop rotation with conservation agriculture (CA) have not been extensively explored thus far in Southern India. Thus, a CA experiment was undertaken to investigate the impact of tillage and weed management on soil microbiome and fungal diversity at 30 days after sowing (DAS) and tasseling stage (60 DAS) of maize (in the 8th crop cycle) and to monitor yield. Three tillage practices; T1:CT(C)-CT(M)-fallow(NSr), T2:CT(C)-ZT(M)-ZT(Sr) and T3:ZT(C) + SrR-ZT(M) + CR-ZT(Sr) + MS and weed control tactics involved; W1-Chemical weed control, W2-Herbicide rotation (in alternative year), W3- Integrated weed management (IWM) and W4-Single hand-weeded control were laid-out in split-plot design. Rhizosphere soil and rhizoplane samples were collected from the respective plots at 30 DAS after herbicides application and tasseling stage (60 DAS). Analysis for microbial population and fungal diversity, enzyme and microbial activities was done duly following standard procedures. The salient findings indicated; a decline in enzyme activities, microbial population, microbial activities at initial stages (30 DAS) due to impact of herbicides which later on increased by tasseling. These biological properties were higher under T3 and W4, followed by IWM except metabolic quotient (qCO2) which showed a decreasing trend relative to T1, T2 and W1, W2 at both sampling stages of maize. Kernel yield (KY) and System yield (SY) were enhanced by T3 and IWM, herbicides treated plots (W1 and W2) compared to T1, T2 and W4. Talaromyces flavus, a beneficially rhizosphere micro-inhabitant was identified under T3 in combination with IWM. Considering both crop productivity and soil biological assessment, T3 and IWM were considered the best treatment combinations among all others with SY (4453 kg ha− 1). These findings signify the importance of adopting reduced tillage (T3) and IWM to achieve sustainable biodiversity and food security. This will further help the farmers and the policy makers to deeply understand the effectiveness of developing sustainable and eco-friendly tillage practice and weed control strategy for improving soil health and bolstering sustained crop productivity.