Glyphosate-based herbicides are commonly used to combat weeds and unwanted grasses in many habitats in the Hawaiian Islands, including near freshwater, marine, and anchialine pond shorelines. Glyphosate is reported to degrade within a few days of application and to break down rapidly in soil, which suggests that it is safe for use near aquatic environments. However, glyphosate can be transported to coastal waters, especially during run-off events. Five native macroalgael and seagrass species and one introduced aquatic vascular plant found in coastal anchialine ponds or in the adjacent intertidal zone were exposed to freshly mixed solutions of a glyphosate-based herbicide in lab experiments. Chlorophyll absorbance and photosystem II (PSII) efficiency were measured after 5 to 7 days of incubation. At herbicide concentrations (0.225 to 1.8 g L−1 glyphosate) below the manufacturer’s lowest recommended concentration (3.6 g L−1 glyphosate), chlorophyll absorbance and PSII efficiency differed significantly from the control (0.0 g L−1 glyphosate). Native macroalgae and seagrasses in marine and anchialine aquatic habitats may be negatively affected by use of glyphosate herbicides to control shoreline weeds.