Microalgae have attracted interests from a range of biotechnology fields due to the variety of valuable bioactive metabolites some species can synthesise. Compounds such as ω3-fatty acids or carotenoid pigments are commercially exploited to provide the materials necessary for product formulations within the pharmacology, nutraceutical or cosmetic sectors. The co-stimulation of several compounds of interest may as such improve the cost-effectiveness of microalgal biorefinery pipelines. This study focused on the microalgal biological model species Phaeodactylum tricornutum (marine diatom) to investigate the effects on lipogenesis and carotenogenesis of combined stressors, here cold temperature shock (from 20 to 10°C) and addition of NaCl salt (5 mg/ml) or the phytohormone abscisic acid (4 mg/l), applied using a two-stage cultivation strategy. Results showed that cold stress with NaCl or phytohormone addition increased the neutral lipid content of the biomass (20 to 35%). These treatments also enhanced the proportions of EPA (22% greater than control) in the fatty acid profile of biomass extracts. In addition, these treatments had a stimulatory effect on carotenogenesis, especially the combination of cold stress with NaCl addition, which returned the highest production of fucoxanthin (33% increase). The gene expression of diacylglycerol acyltransferase (DGAT) and the ω-3 desaturase precursor (PTD15), as determined by real time PCR, were enhanced 4- and 16-fold relative to the control, respectively. In addition, zeaxanthin epoxidase 3 (ZEP3), involved in the xanthophyll cycle, was downregulated at low temperature when combined with abscisic acid. These results highlight the benefits of applying a combination of low temperature and salinity stress, as part of a two-stage cultivation process, to simultaneously enhance the yields of the valuable metabolites EPA and fucoxanthin in Phaeodactylum tricornutum.