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Grassland management and climate change may have profound effects on soil organic carbon (SOC) storage in temperate grasslands, but to date only limited information on the long-term SOC development derived from grassland monitoring sites is available. In 1986, the Bavarian State Research Center for Agriculture established 20 long-term grassland soil monitoring sites in the state of Bavaria, Germany. We analyzed SOC changes and their main controlling factors in these monitoring sites between 1989 and 2016 in four intervals using the machine learning algorithm conditional random forest. The results showed that changes of SOC stocks in grassland monitoring sites were affected by the multifactorial and interactive nature of changing climate conditions, pedogenic-topographic factors and management practices. Seasonal climate variables explained the highest variability of SOC stock changes in long-term grassland sites, followed by organic fertilizer application. Increasing autumn precipitation led to decreased SOC stocks, whereas increasing spring and summer precipitation led to increased SOC stocks on sites at high elevations with low slopes. Furthermore, we observed that the organic fertilizer application resulted in an increase and/or preservation of SOC stocks, whereas conventional farming with a low degree of organic fertilization resulted in SOC stock decreases. Regarding the acceleration of climate change and the complex interactions between site conditions, changing climate and management, there is an urgent need to collect improved management data in future long-term studies.