Sulfur (S) is widely used in agriculture, yet little is known about its fates within upland watersheds, particularly in combination with disturbances like wildfire. Our study examined the effects of land use and wildfire on the biogeochemical “fingerprints,” or the quantity and chemical composition, of S and carbon (C). We conducted our research within the Napa River Watershed, California, U.S., where high S applications to vineyards are common, and ~ 20% of the watershed burned in October 2017, introducing a disturbance now common across the warmer, drier Western U.S. We used a laboratory rainfall experiment to compare unburned and low severity burned vineyard and grassland soils. We then sampled streams draining sub-catchments with differing land use and degrees of burn and burn severity to understand combined effects at broader spatial scales. Before the laboratory experiment, vineyard soils had 2–3.5 times more S than grassland soils, while burned soils—regardless of land use—had 1.5–2 times more C than unburned soils. During the laboratory experiment, vineyard soil leachates had 16–20 times more S than grassland leachates, whereas leachate C was more variable across land use and burn soil types. Unburned and burned vineyard soils leached S with δ³⁴S values enriched 6–15‰ relative to grassland soils, likely due to microbial S processes within vineyard soils. Streams draining vineyards also had the fingerprint of agricultural S, with ~2–5 fold higher S concentrations and ~ 10‰ enriched δ³⁴S-SO₄²⁻ values relative to streams draining non-agricultural areas. However, streams draining a higher fraction of burned non-agricultural areas also had enriched δ³⁴S values relative to unburned non-agricultural areas, which we attribute to loss of ³²S during combustion. Our findings illustrate the interacting effects of wildfire and land use on watershed S and C cycling—a new consideration under a changing climate, with significant implications for ecosystem function and human health.