Microalgae are commonly referred to as ‘unicellular factories’ due to their size and their capacity to produce and accumulate valuable biomolecules. The synthesis of a given biomolecule is generally triggered by external stimuli (e.g., light). This work evaluated the effect of different types of water and nutrient sources (freshwater, wastewater, fertilisers, or pig slurry) on the biochemical composition of the microalga Tetradesmusalmeriensis produced on a pilot-scale. Overall, significant differences were observed in the biochemical composition of the biomasses (p < 0.05). The biomass produced using freshwater and fertilisers showed potential to be used as food given its high content of essential amino acids, carotenoids (mainly lutein and β-carotene), and PUFAs (C18:2 and C18:3). The microalgae produced using wastewater showed potential applications in the animal feed and agricultural industries. They showed a higher abundance of essential amino acids and carotenoids, including fucoxanthin and lycopene; the latter was only detected when the biomass was produced using secondary wastewater. The biomass produced using pig slurry as the source of nutrients showed potential for being used as animal feed or as an agricultural product given its high content in essential biomolecules and amino acids. The biochemical composition was not only a cause of different microbial diversities but also of the effect of the different composition of the culture medium that trigger different responses of the microalgal cells.