Microalgae as feedstock for biofuel production have attracted serious consideration as an important sustainable source of energy. For biodiesel production with microalgae, a series of consecutive processes should be performed as selection of adequate microalgal strains, mass culture, cell harvesting, oil extraction and transesterification. The aim of this study was to investigate the growth and lipid accumulation of a new isolated marine microalgal strain by optimizing culture medium composition and applying different stressful culture conditions. Microalga CTM 20023 was isolated from the evaporating salt-ponds at Sfax, Tunisia, using serial-dilution technique from enriched cultures. Phylogenetic analysis based on SSU rDNA and rbcL-3P sequences attributed this isolate to a new species of the Amphora genus. This wild strain possesses rapid gravity sedimentation of 2.91 m h(-1), suitable for an easy and low-cost biomass harvest. The optimization of the composition of the culture medium through statistical experimental designs improved the specific growth rate of Amphora sp. from 0.149 to 0.262 day(-1) and increased its 15-day culture biomass production from 465 to 2200 mg L(-1) (dw) and its lipid content from 140 to 370 mg g(-1) (dw). Highest biomass productivity of 178 mg L(-1) day(-1) was achieved at the 10th day of culture. Highest lipid content of 530 mg g(-1) (dw) was obtained under phosphorus starvation and 64.34% of these lipids were saturated fatty acids. A first growth stage, in optimized condition, would thus offer the maximum productivity for an algal biomass feed stream, followed by second stressful stage for lipid accumulation, thus suitable for biodiesel production.