Pulse amplitude modulation (PAM) fluorometry is ideally suited to measure the sub-lethal impacts of photosystem II (PSII)-inhibiting herbicides on microalgae, but key relationships between effective quantum yield [Y(II)] and the traditional endpoints growth rate (μ) and biomass increase are unknown. The effects of three PSII–inhibiting herbicides; diuron, hexazinone and atrazine, were examined on two tropical benthic microalgae; Navicula sp. (Heterokontophyta) and Nephroselmis pyriformis (Chlorophyta). The relationships between Y(II), μ and biomass increase were consistent (r2 ⩾ 0.90) and linear (1:1), validating the utility of PAM fluorometry as a rapid and reliable technique to measure sub-lethal toxicity thresholds of PSII-inhibiting herbicides in these microalgae. The order of toxicity (EC50 range) was: diuron (16–33 nM) > hexazinone (25–110 nM) > atrazine (130–620 nm) for both algal species. Growth rate and photosynthesis were affected at diuron concentrations that have been detected in coastal areas of the Great Barrier Reef.