Recycling of livestock manure in agroecosystems has been shown to enhance the sustainability of food production and reduce adverse environmental consequences from intensive crop-livestock systems. However, the effect of manure application on the associations between soil quality and ecosystem multifunctionality still remains poorly understood. Hereby, we used a five-year field experiment to investigate the effect of mineral and manure fertilization on soil quality, enzymatic stoichiometry, and ecosystem multifunctionality for both topsoil and subsoil (i.e. 0-20 cm and 20–40 cm). Manure alone and combined with mineral fertilization increased soil quality index by 49.5% and 70.1% in the topsoil, and by 67.5% and 26.6% in subsoil compared to no fertilization. Moreover, the manure application increased the C, N, and P acquisition enzyme activities, especially those for C and P cycling. Fertilization regimes affect enzymatic stoichiometry in the subsoil rather than topsoil. Manure application increased soil ecosystem multifunctionality in both top and subsoil by 2.1 and 0.4 times, respectively. Interestingly, the soil quality index was positively correlated with ecosystem multifunctionality regardless of fertilization regimes. Furthermore, random forest analysis showed that soil organic C and N content, available P, and microbial biomass were the main drivers of soil ecosystem multifunctionality. Conversely, mineral fertilization did not affect soil quality and enzyme activity in both soil layers, and thus did not change soil ecosystem multifunctionality. In conclusion, manure application fosters soil quality and has the potential to improve the soil multifunctionality, thereby providing an effective way to sustainable soil management and cleaner crop production.