Water quality along the 20 m depth isopleth of Lake Ontario was assessed using continuous sampling at a depth of 1.5 m by drawing water through the hull of a ship into a ferry box containing sensors. Water temperature, specific conductivity, chromophoric dissolved organic matter (CDOM), and phytoplankton groupings (by pigment-specific fluorometry) were monitored continuously over the 4-day route and water was collected for determination of size-fractionated extracted chlorophyll a (Chl a) concentration at hourly intervals. Total (> 0.2 μm) extracted Chl a ranged from 1.91 to 6.32 μg/L with an average (± SD) of 3.19 ± 0.87 μg/L: picoplankton (0.2−2 μm) represented the greatest contribution (43 ± 11%) to the Chl a, followed by the nanoplankton (2–20 μm; 31 ± 9%), and the microplankton (> 20 μm; 26 ± 8%). Among the phytoplankton, the Heterokontophyta and Pyrrophyta represented the majority (61.4%) of the in situ total Chl a measured along the entire 20 m isopleth, followed by the phycoerythrin-rich Cyanobacteria and Cryptophyta group (18.2%), the phycocyanin-rich Cyanobacteria group (17.8%), and the Chlorophyta and Euglenophyta group (2.6%). Peaks of phytoplankton in the surface water corresponded to nearshore water adjacent to known sources of high phytoplankton (e.g. tributaries) and were associated with detectable changes in water quality, as indicated by CDOM, temperature, and specific conductivity. This investigative survey demonstrates the utility of using a ferry box for continuous water quality measurements in the nearshore environment of the Great Lakes and opens further applications for wide-scale assessment of watershed influences on nearshore water quality.