Antimicrobial resistance is a growing global issue, driven in part by the mass consumption of antibiotics. Moreover, most antibiotics are used in animal husbandry for the prevention/treatment of livestock infections, and are excreted largely untransformed, exerting selective pressure on bacteria in livestock feces. Aged manure is often applied to farm fields as a soil amendment but can enter adjacent streams during storm runoff. In this way, genes encoding resistance to antibiotics (i.e., antimicrobial resistance genes; ARGs) and antibiotic residues may exert pressure on environmental bacteria and contribute to the spread of antibiotic resistance (AR). Currently, there are gaps in our understanding of the mechanisms driving AR, including the impact of nutrients, light, and biofilms on the removal of ARGs from streams. To examine controls on water column removal of three ARGs (tetracycline-resistant tetW and tetQ and erythromycin resistant ermB), we released ARG-rich manure slurry into 24 recirculating mesocosms, using a fully-crossed design; experimental treatments included shaded versus unshaded mesocosms, elevated versus ambient nutrients, and biofilm-colonized versus uncolonized substrate. Removal rates (k, expressed in h-1) for tetQ ranged from k=0.01-0.3 h-1, the highest removal rate occurred with biofilm, under light conditions, with added nutrients. Removal rates for tetW were similar to tetQ, and ranged from k=0.01-0.27 h-1, while ermB removal rates showed the widest range across targets, varying from k=0.03-0.29 h-1. Overall, biofilm colonization increased removal rates for all targets (General Linear Model; P<0.001 for all). Light availability, which mediated biofilm colonization, also increased removal rates (GLM; P<0.05 for all). Finally, the addition of nutrients on ARG removal was weaker and more variable; nutrients did not alter tetW removal rates, moderately impacted tetQ removal rates (P=0.05), and strongly influenced ermB removal (P<0.01). Our research highlights that biofilms, which are mediated by light and nutrients, can influence ARG removal from streams.