Trends in soil organic carbon (SOC) were analyzed in the soils from the oldest Czech long-term field experiment, the Prague-Ruzyně Long-Term Fertilizer Experiment, conducted on Haplic Luvisol since 1955. The aim of the work was to compare the long-term dynamics of SOC in contrasting crop rotations and different fertilization regimes. The trial design includes two crop rotations (CR): simple CR with two-year rotation of sugar beet and spring wheat, and multi-crop rotation (MCR) with nine crops. Four fertilization treatments were chosen for SOC analysis: unfertilized control, only mineral fertilization (NPK), farmyard manure application (FYM), as well as FYM and NPK application. SOC content was significantly affected by both fertilization and crop rotation practices. In the simple CR, both the unfertilized control and the NPK treatment exhibited a consistent decline in SOC content over the study period, with percentages decreasing from an initial 1.33% in 1955 to 1.15% and 1.14%, respectively. Although the FYM and FYM + NPK treatments showed an increase in SOC content in the 1990s, a gradual decline was recorded in the last two decades. This decrease was not observed in MCR: positive C balances were recorded in all treatments within MCR, with the largest increase in SOC stock occurring when NPK was combined with FYM. In contrast, over the last decade, C balances have decreased in simple CR for all treatments except FYM. This trend coincides with changes in the local climate, particularly rising temperatures. The results indicate that diversified crop rotations and FYM fertilization are effective in mitigating the negative impacts of changing environmental conditions on SOC stocks.