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We examined the effect of agricultural land use on whole-stream nutrient availability, biofilm standing crop, and biofilm nutrient limitation and uptake in 21 stream locations in southeastern Idaho. Higher stream water concentrations of dissolved organic carbon (DOC), but not nitrate (NO3-N) or phosphate (PO4-P), were associated with % agriculture in the watershed. Streambed chlorophyll a (Chl a) and ash free dry weight (AF dry wt) also increased with agricultural land use. Nutrient diffusing substrate (NDS) bioassays, which determine biofilm nutrient limitation, showed that Chl a was NO3-N limited and suppressed by labile DOC, whereas AF dry wt did not respond to either NO3-N or labile DOC additions. Together, the different responses of Chl a and AF dry wt suggest potential competition between biofilm autotrophs and heterotrophs for nutrients or other resources. Nutrient uptake, determined with short-term releases of NO3-N, PO4-P, and DOC (as glucose) showed that NO3-N uptake velocity (V-f) increased with agricultural land use. Despite the N-limited status of biofilms indicated by the NDS results, uptake of NO3-N could not be consistently detected at all locations. The differences in response of biofilm to organic carbon enrichment suggests a difference in DOC quality, with labile DOC added with NDS compared to perhaps less-labile DOC found in-stream. Linking the interactive responses of biofilm communities to altered nutrient availability is an important step toward understanding whole ecosystem responses to land-use change.