The demand for functional food is surging worldwide, reflecting the developing awareness of the role of food in maintaining good health. It is crucial to ensure that diets are rich in bioactive properties to help prevent and treat chronic diseases. Limnospira (Spirulina) is a filamentous edible cyanobacterium with significant potential as a functional food. It contains various bioactive compounds, including proteins, carbohydrates, and pigments. Additionally, it can produce essential polyunsaturated fatty acids (PUFAs) such as linoleic and γ-linolenic acid. In this study, we analyzed the fatty acid compositions, particularly the production of γ-linolenic acid, in six strains of Limnospira fusiformis cultivated with phosphorus-limited anaerobic digestion effluent (ADE) derived from water hyacinth, as well as in standard algal media. Even at 8-fold diluted ADE, which contained a limited amount of phosphate, the algal biomass production reached 0.63 g L⁻¹. Except for the La-08 strain, all other strains produced a higher amount of PUFAs and exhibited lower chlorophyll content when grown in the 8-fold diluted ADE compared to those reared in standard algal media. This finding suggests that cultivating Limnospira using ADE from the overgrowth of water hyacinth has a great potential for producing valuable biomolecules, such as γ-linolenic acid, through sustainable nutrient recycling.