Artificial light at night (ALAN) is pervasive in urban environments, yet its potential effects on biotic communities of urban streams are not fully understood. We investigated the effects of ALAN on fish assemblages in 20 lit-unlit paired stream reaches across 10 sites in Columbus, Ohio, USA across two years. Our results show that light trespass into urban streams is approximately doubled during leaf-off in winter months (1.97 lux ± 0.28) compared to leaf-on in summer months (1.00 lux ± 0.19), highlighting the important interceptive effect of canopy coverage in headwater systems. There were no differences in fish assemblages (i.e., abundance, relative abundance, biomass, diversity, guild composition) between lit and unlit reaches (> 100 m apart). Species richness and α-diversity (Hill numbers) scaled positively with mean stream depth. Nutrient concentrations were negatively related to mean fish mass and fish assemblage diversity. Species richness scaled negatively with total phosphorus and α-diversity scaled negatively with orthophosphate. Our results imply that the relatively low-levels of ALAN observed in canopied streams may exert only subtle effects on small stream fish assemblages, which are difficult to detect amongst the multiple, confounded stressors impacting these relatively species-poor, urban systems. Experimental approaches or large-scale manipulative studies may be necessary to isolate the effects of ALAN on streams and fish in urban contexts.