Update information. Antibiotics are increasingly detected in aquatic environments, and their potential ecological risk is a concern. However, most antibiotic toxicity studies were performed with single exposure experiments. Here, we studied the effects and mechanisms of repeated clarithromycin (CLA) exposure on the algae Microcystis aeruginosa compared to the results of a single exposure. The 96-h effective concentration of CLA was 13.37 μg/L upon single exposure but was reduced to 6.90 μg/L upon repeated exposure. Single-exposure CLA inhibited algal photosynthesis by disrupting energy absorption, dissipation and trapping, reaction center activation, and electron transport, thereby inducing oxidative stress and ultrastructural damage. CLA also upregulated glycolysis, pyruvate metabolism, and the tricarboxylic acid cycle. Stronger inhibition of algal growth was observed under repeated exposure via effects on photosynthetic pigments, biosynthesis of reaction center subunits, and electron transport, inducing more substantial oxidative damage. Furthermore, repeated exposure reduced carbohydrate utilization by blocking the pentose phosphate pathway, which changed the characteristics of extracellular polymeric substances and eventually impaired algal self-defense. Risk quotients calculated from repeated exposure were higher than 1, indicating significant ecological risks. This study elucidated the severe influence of repeated antibiotic exposure on algae, providing new insights into antibiotic risk assessment.