The rapid rise in atmospheric CO₂ necessitates strategies for mitigation and valorization. Microalgae offer potential through simultaneous CO₂ capture and production of high-value biomolecules. Five Chlorophyta strains (A–E: Micractinium sp., Chlamydomonas sp., Micractinium sp., Chlorococcum sp., and Chlorella vulgaris) were isolated from temperate waters and soils and tested for growth and biochemical responses under controlled nitrogen availability (low: 0.346 g L⁻¹ nitrate; high: 0.6 g L⁻¹ nitrate + ammonia), carbon supply (low: 0.04% CO₂; high: 4% CO₂), and cultivation systems (batch reactors, fermenters, and varied illumination). Over 14 days, maximum dry biomass was achieved in batch cultivation with CO₂ sparging, low nitrogen, and continuous light, ranging from 1.47 g L⁻¹ (strain A) to 2.67 g L⁻¹ (strain D). Biomass composition varied: proteins, 25–45%; carbohydrates, 20–35%; and lipids, 18–28%. Nitrogen limitation promoted lipid accumulation (e.g., strain D: +40%) with concurrent protein decline (−25%). Chlorophyll a/b displayed strain-specific plasticity; high CO₂ generally increased chlorophyll, while nitrogen stress reduced it up to 50%. Overall, this study demonstrates that locally adapted Chlorophyta strains can achieve high biomass productivity under CO₂ enrichment while allowing for flexible redirection of carbon flux toward lipids, carbohydrates, or pigments through nutrient management. Among the tested isolates, strains D and E emerged as the most promising candidates for integrated CO₂ sequestration and biomass production, while strains B, C, and D showed strong potential for biodiesel feedstock; strain A for carbohydrate valorization; and strain E for chlorophyll extraction. Future research should focus on scale-up validation in pilot photobioreactors under continuous operation, optimization of two-stage cultivation strategies for lipid production, integration with industrial CO₂ point sources, and strain improvement using modern genomics-assisted breeding and genome-editing technologies. These efforts will support the translation of regional microalgal resources into scalable carbon-capture and bioproduct platforms.