Nitrogen-fixing cyanobacteria are vital photosynthetic microorganisms that contribute to soil fertility by fixing atmospheric nitrogen and are also important for maintaining ecosystem stability. These microorganisms can be very sensitive to herbicides because they possess many characteristics of higher plants. Six days after the application of monosulfuron at 0.03 to 0.3 nmol L−1 under laboratory conditions, growth of the nitrogen-fixing cyanobacteria Anabaena flos-aquae, Anabaena azollae, and Anabaena azotica was stimulated, but at higher concentrations (30 to 300 nmol L−1) protein synthesis was inhibited. The production of 16 amino acids in A. flos-aquae was reduced from 7 to 69% with increasing monosulfuron concentration. Application of monosulfuron at 3 to 300 nmol L−1 substantially inhibited in vitro acetolactate synthase (ALS) activity as indicated by 50% inhibition index values of 3.3, 65.2, and 101.3 nmol L−1 for A. flos-aquae, A. azollae, and A. azotica, respectively. In contrast, extractable ALS activity was not affected in these algal species with monosulfuron treatments ranging from 0.03 to 300 nmol L1 except in A. flos-aquae at higher concentrations (30 to 300 nmol L−1). The most sensitive species to monosulfuron was A. flos-aquae, followed by A. azollae and A. azotica. Molecular analyses showed that the genomic DNA of A. azollae and A. azotica differed in only one amino acid. Results from photogenetic analyses revealed a high degree of homology between these algae. In contrast, the genomic DNA of A. flos-aquae differed from that of A. azollae and A. azotica in 44 and 45 amino acids, respectively. Our findings support the view that monosulfuron toxicity in these three nitrogen-fixing cyanobacteria is due to interference with protein metabolism via inhibition of branch-chain amino acid biosynthesis, and particularly ALS activity.