Three cyanobacterial strains of the genus Spirulina, i.e., LAMB171, LAMB172, and LAMB220, were cultivated at different CO2 concentrations in a columnar photobioreactor to determine the most optimal strain for CO2 mitigation and co-production of bioactive substances, specifically carotenoids, C-phycocyanin and allophycocyanin (A-PC). The best biomass production and the highest CO2 fixation rate were obtained at 10% CO2 concentration for all three Spirulina strains evaluated in the present study. When compared with the control group, the contents of chlorophyll a, carotenoids, C-PC, and A-PC were more optimal in the investigated Spirulina strains at lower CO2 concentrations (2% and 5%). However, the content of these bioactive substances was not significantly different at a 10% CO2 concentration compared with that at lower CO2 concentrations. Overall, it was determined that 10% CO2 concentration was noted to be most suitable for the production of these bioactive substances owing to the highest biomass productivity for all three Spirulina strains. Besides, LAMB220 was found to be the most effectively mitigated CO2 in three strains on account of its high biomass productivity and excellent C-fixation capability, while LAMB171 and LAMB172 species were more suitable for the production of pigments at a 10% CO2 concentration. •Three Spirulina strains were cultivated at different CO2 concentrations in a columnar photobioreactor.•The highest CO2 fixation rate was found at 10% CO2 concentration for all three strains.•10% CO2 concentration was the most suitable for production of the bioactive substances.•LAMB220 was the species most effectively mitigated CO2.•LAMB171 and 172 were more suitable for the production of pigments at 10% CO2 concentration.