This study aimed to evaluate the potential of six oleaginous eustigmatophytes for use as biorefinery feedstock for the co-production of palmitoleic acid (PA) and biodiesel under different initial nitrogen concentrations (INCs). Six eustigmatophytes were studied, the nitrogen deficiency strategy significantly stimulated the simultaneous hyper-accumulation of PA and lipids, and led to a desirable fatty acid profile (FAP), except in Vacuoliviride sp. and Nannochloropsis oculata. Particularly, Eustigmatos cf. polyphem exhibited great potential when supplied with 1 mM INC and yielded the highest PA (29.71% of dry weight (DW)) and lipid (72.01% of DW) contents, as their productivities increased to 96.26 and 232.79 mg/L/d, respectively. Furthermore, neutral lipids accounted for 91.82% of the total lipids and were rich in PA, and the favourable FAPs of C16-C18 (87.95%) and monounsaturated FAs (70.10%) ensured good biodiesel properties including the cetane number (55.69) and iodine value (92.81 gI(2)/100 g), and all met the standard requirements.