Since invading the Laurentian Great Lakes in the late 1980s, round goby Neogobius melanostomus have become a dominant benthic prey species, resulting in a need to accurately monitor their population abundance to inform fisheries management. Camera-based methods for assessing round goby abundances have gained popularity, but their efficiencies for detecting round goby are poorly understood. We evaluated the efficiency of down-looking color monocular cameras for detecting round goby presence in microcosm enclosures with known numbers of fish. Detection efficiencies were compared between sand and cobble substrate during daylight hours using a generalized linear mixed-effects model. After accounting for trial effects, enclosure placement, and within-replicate temporal autocorrelation, round goby detection efficiency was 98.6% over sand (95% confidence limit (CL): 98.0–99.0%), and 55.3% over a single layer of cobble substrate (CL: 48.6–61.8%). The large difference in detectability between cobble and sand suggests that camera-based estimates of round goby abundances are likely to be biased low and have lower relative precision in high-structure habitats, but may be largely unbiased in low-structure habitats. Despite much lower relative detection efficiencies of cameras in high-structure cobble habitats, the efficiencies reported here still compare favorably to conventional methods like otter and beam trawling. Our results provide initial quantitative bounds on the possible degree of negative bias in camera-based estimates of round goby abundances in different substrates, but require further field verification across the diversity of substrates, interstitial infilling, and other structural attributes of habitat found in the Great Lakes.