Knowledge of the origin of sedimentary organic matter (SOM) in lakes contributes to our understanding of the carbon cycle and provides valuable guidance for lake ecosystem management. To elucidate the changes in the spatial and seasonal origins of SOM in a eutrophic lake, we collected sediment at 31 sampling points in Lake Hachiro, northern Japan, in June and November of 2016 and April and June of 2017, and measured total organic carbon (TOC) and fatty acid biomarkers. We found that TOC and the origin of SOM showed both spatial and seasonal variations. TOC was relatively high in the eastern part of the lake and may reflect the hydrological conditions therein. The average TOC of sediment collected from the 31 sampling points increased from June to November 2016, concomitant with an increase in cyanobacterial fatty acids. A cyanobacterial bloom was observed in the water column from July to October of that year, and the input of cyanobacterial carbon to the sediment may explain the TOC increase during this period. We also detected an increase in TOC from November 2016 to April 2017, which coincided with increases in fatty acids of terrestrial and diatom origin. In this regard, we suspect that meltwater inflow during this period may have borne large quantities of terrestrial organic matter, and diatoms are known to predominate in Lake Hachiro during the spring. In contrast, from April to June 2017, we recorded decreases in TOC and all fatty acid biomarkers, with the exception of those derived from bacteria, indicating that decomposition processes prevailed during this period. Quantitative estimates indicated that, despite the spatial and seasonal variations, terrestrial organic carbon was the predominant carbon source in Lake Hachiro, even when the waters were eutrophic and algal blooms were present.