Euglena sp. is a freshwater microalga that produces useful metabolites in its biomass. The cultivation with oxidative stress treatments, such as salinity, plays a major role in maintaining the optimal cellular metabolic rate for the optimized growth of Euglena sp. for the production of lipids for biodiesel as well as carotenoids and chlorophyll, which are cell defense pigments that are beneficial for health. A bioflocculation method that uses microalgal flocculants, such as Skeletonema sp., is an alternative harvesting technique that is cost and energy saving. The effect of salinity and bioflocculation treatment on freshwater microalgae has not been widely studied. Therefore, this research determined the effect mechanism of salinity and bioflocculation in the production of lipids, chlorophyll, and carotenoids in Euglena sp. with Skeletonema sp. as a bioflocculant. In this research, the cultivation of Euglena sp. was carried out in Cramer–Myers medium for seven days under salinity treatments of 5, 10, 20, and 0 g/L, and the cultivation of Skeletonema sp. was performed for eight days in F/2 medium with modified silicate removal. Bioflocculation was performed by mixing Euglena sp. and Skeletonema sp. at ratios of 1:1, 1:0.5, and 1:0.25. The research results showed that salinity treatment, in general, succeeded in increasing the growth and production of lipid, chlorophyll, and carotenoid metabolites. The addition of Skeletonema sp. to the culture of Euglena sp. increased the precipitation percentage. A high mixing ratio increased the lipid level but decreased those of chlorophyll and carotenoid metabolites.