To assess the potential impact of agricultural activity on a stream fish in its natural habitat, we examined the relative brain size of creek chub (Semotilus atromaculatus) from fourteen different stream sites of Southwest Ontario, Canada, in relation to a comprehensive set of physical, water quality, and ecological variables. Creek chub showed broad variation in mean relative brain size (corrected for body size) between these sites chosen to represent a gradient of agricultural activity. Despite this variation, there was no correlation between measures of agricultural activity and relative brain size. Although 17 different pesticides were detected across sites, no discernible associations of these compounds and relative brain size were seen. Higher summer water temperatures were associated with larger relative brain size. Additionally, the proportion of water quality-sensitive stream insect larvae, itself positively correlated with dissolved oxygen levels, was positively associated with relative brain size but not with water temperature. The study reveals that creek chub relative brain size is more closely linked to environmental and ecological parameters than conventional indices of agricultural activity in a region dominated by agriculture. These findings highlight the multifactorial influences contributing to brain growth in wild fishes and emphasize the difficulties in extrapolating neurotoxic effects observed in laboratory studies to natural systems.