Natural carotenoids from green microalgae exhibit beneficial effects in treating health-related diseases, primarily due to their antioxidant capacity. Therefore, carotenoid-producing microalgae were isolated and characterized from aqueous samples in KwaZulu-Natal (South Africa) under photoautotrophic conditions. Three isolates were characterized comprehensively using light and electron microscopy. In addition, the growth of the three selected microalgae was evaluated under photoautotrophic and photoheterotrophic conditions. Based on the cytological characteristics, the three strains matched the genera Haematococcus, Parachlorella, and Scenedesmus, the latter two additionally confirmed via analysis of the 18S rRNA gene sequence at the genus level. Light and electron microscopy and UV-Vis spectroscopy confirmed pigment production by all three microalgal strains. Both isolate Parachlorella sp. AA1 and Scenedesmus sp. AA2 showed the highest growth rate when cultured with acetate (25 mM) photoheterotrophically, while the isolate Haematococcus sp. AA3 grew best with glucose (50 mM). In addition, all three isolates utilized salicylate as a carbon source under photoheterotrophic conditions, evidently resulting in increased carotenoid production in strains AA1 and AA3. The 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) antioxidant assay confirmed that methanol extracts of all three isolates contained carotenoids that can scavenge radicals, and thin layer chromatography (TLC) analysis showed that β-carotene and astaxanthin were formed by strain AA3 as main carotenoids.