Plant-derived allelochemicals have the potential to be used as eco-friendly measures for control of Microcystis aeruginosa blooms. However, the susceptibility of M. aeruginosa to plant allelochemicals under a range of environmental factors, such as alkalinity, is not clear. This study investigated the effects of different alkalinity treatments on 1,2,3-trihydroxybenzene (pyrogallol) toxicity to a toxic strain of the cyanobacterium, M. aeruginosa (FACHB 905). Pyrogallol toxicity to M. aeruginosa cultures, both pre-adapted to a range of alkalinity levels and un-adapted, increased when alkalinity was increased from 0.09 to 1.51 meq L−1. The mean inhibition ratios calculated according to OD650, cell concentration, Chl a, and carotenoid concentrations of M. aeruginosa cultures were highest in the highest alkalinity treatment (1.51 meq L−1), i.e., up to 74%, 80%, 73%, and 87% for alkalinity-adapted cells on day 3. The lowest cell concentrations and photosynthesis pigment concentrations were found in the highest alkalinity treatment (1.51 meq L−1) for un-adapted cells in 12-day bioassays. This trend was more obvious over time. Pyrogallol and TPC (total phenolic compounds) concentrations measured immediately after pyrogallol addition into the culture medium decreased more rapidly in higher alkalinity treatments. In contrast, faster oxygen consumption and higher production of quinone end products occurred within the first 30 min after pyrogallol addition at higher alkalinity levels. Quinones and oxygen radicals have been shown previously to be more toxic to cyanobacteria than pyrogallol itself. This provides a potential explanation for the enhanced pyrogallol toxicity to M. aeruginosa under higher alkalinity. Therefore, it is important to take alkalinity into account when considering pyrogallol as a potential biocide.