This work aimed to characterize two native microalgal strains newly isolated from South Mediterranean areas and identified as Chlorella sorokiniana ES3 and Neochloris sp. AM2. The growth properties and biochemical composition of these microalgae were evaluated in different culture media (Algal, BG-11, f/2, and Conway). Among the tested media, nitrate- and phosphate-rich Algal medium provided the maximum biomass productivities (85.5 and 111.5 mg l(-1) day(-1) for C. sorokiniana and Neochloris sp., respectively), while the nitrate- and phosphate-deficient f/2 medium resulted in the highest lipid productivities (24.1 and 35.8 mg l(-1) day(-1) for C. sorokiniana and Neochloris sp., respectively). The physiological state of both microalgae was investigated under different light and temperature levels using the pulse amplitude-modulated fluorometry. The better photosynthetic efficiency of C. sorokiniana was obtained at 23 A degrees C with a light saturation of 156 mu E m(-2) s(-1), while that of Neochloris sp. was achieved at 15 A degrees C with a light saturation of 151 mu E m(-2) s(-1). The analysis of fatty acid profile and biodiesel parameters revealed that C. sorokiniana, cultivated in Algal and f/2 media, can be considered as a suitable candidate for high-quality biodiesel production.