To investigate the occurrence of UV sunscreening biomolecules and their role in photoprotection in cyanobacterial biofilms growing in brightly lit habitats with high UV fluxes. High performance liquid chromatography with photodiode-array and mass spectrometry revealed the presence of mycosporine-like amino acids (MAAs) shinorine (λ(max) 334 nm, m/z 333), porphyra-334 (λ(max) 334 nm, m/z 347), mycosporine-glycine (λ(max) 310 nm, m/z 246) and palythinol (λ(max) 332 nm, m/z 303). Two unknown MAAs with λ(max) at 320 (m/z 289) and 329 nm (m/z 318) were also found. Biosynthesis of MAAs was found to increase with increase in exposure time under UV radiation. The MAAs from biofilms showed efficient radical scavenging activity as well as photoprotective potential on the survival of UV-treated Escherichia coli cells. Biosynthesis of photoprotectants is an important mechanism to prevent photodamage in Cyanobacteria. UV-induction and photoprotective function of MAAs may facilitate them to perform important ecological functions under harsh environmental conditions. There are very few reports on qualitative and quantitative characterization of different MAAs in cyanobacterial biofilms. Due to strong UV absorption and photoprotective function, MAAs may be used as an active ingredient in cosmetic and other pharmaceutical industries.