Throughout the Mediterranean biome, fire has been a dominant natural agent of change and an important tool for anthropogenic landscape modifications for millennia. However, fundamental knowledge on the complex linkages among fire, vegetation, and early agricultural practices remains limited. This research explores the role these processes have in shaping the landscape in the southern Levant – a key region with a long tradition of agriculture and horticulture. Olive horticulture is among the oldest and most widespread agricultural forms in the Mediterranean Basin. The earliest palynological evidence for olive horticulture is recorded in the Sea of Galilee in Israel and suggests that the first major olive cultivation began during the end of the 8th millennium BP. We present a multi-proxy dataset from the Sea of Galilee before and during the first olive cultivation (~7700–6680 a cal BP). Quantitative analyses of a charcoal time series categorized into distinct charcoal morphotypes indicate changes in fire regimes and sources of biomass burning. With a two-year-resolution, our study offers one of the highest resolution sedimentary charcoal records yet available worldwide. Isotopes from lake carbonates provide new evidence for the climatic setting of this phase, providing an independent evaluation of climatic impacts on vegetation and fire. Combined with palynological evidence, this multi-proxy dataset provides new insights into the linkages between fire, climate, fuel, and early horticultural practice. The fire regime changed with the start of olive cultivation in the Sea of Galilee's catchment. Fire frequency increased just before and during the expansion of olive cultivation, probably driven by the anthropogenic use of fire for clearing the landscape and promoting fertile soils. Once olive orchards were established, low magnitude fires became common, suggesting anthropogenic manipulation of the fire regime with frequent, low intensity, controlled burnings. Fuel limitation and low fuel connectivity likely played a role in both pre-cultivation and cultivation phases, resulting in low fire activity during times of reduced grass and woody vegetation cover. This study allows a new perspective into the temporal variability of middle-Holocene fire regimes in the Levant and an increased understanding of the dynamic role of fire during early horticulture practices. Our data also have relevance for future challenges that cultural legacies such as olive cultivation will face.