Intracellular free Ca²⁺, [Ca²⁺]_i, is a key element of the cellular response to many abiotic and biotic stresses. Cyanobacteria are ancient organisms which have evolved signal transduction systems in response to an ever changing environment. Cyanobacteria have been found to respond to specific and quick Ca²⁺ signatures (unique combinations of changes in [Ca²⁺]_i) when exposed to environmental stress. Antibiotics are bioactive molecules which are being considered as pseudopersistent contaminants as they are being continuously released to the environment; although non-target organisms, cyanobacteria as prokaryotes are sensitive to antibiotics. In order to check whether [Ca²⁺]_i could be involved in sensing antibiotic exposure, we have recorded the Ca²⁺ signatures induced by antibiotics belonging to different classes at two effective toxicity concentrations, the EC₁₀ and the EC₅₀. For that, we have used a recombinant bioluminescent strain of the cyanobacterium Anabaena sp. PCC7120 that constitutively expresses the calcium-binding indicator protein, apoaequorin. We found that Ca²⁺ signatures upon exposure to antibiotics were induced quickly (2–3 seconds) and that they differed between antibiotics and antibiotic concentration. As different classes of antibiotics have been detected simultaneously in the aquatic environment, we also recorded and analyzed the Ca²⁺ signatures induced by binary mixtures and a multiantibiotic mixture; we found that these Ca²⁺ signatures could determine the nature of the interaction between antibiotics in the mixture, whether synergistic or antagonistic, well before toxicity was evident. To our knowledge, this study is the first report of intracellular free Ca²⁺ as an early biomarker of antibiotic exposure in the environment.