A 12-week feeding trial examined the dietary impact of replacing fishmeal (FM) with algal biomass (AB) derived from Pavlova sp. strain CCMP459 (Pav459) in Atlantic salmon diets. Three distinct diets were formulated: a control diet featuring 20% FM and 7% fish oil (FO), an experimental diet incorporating a 50:50 blend of FM and AB Pav459 and reduced FO (10% FM; 4.5% FO; 10% AB), and a second experimental diet with full replacement of FM with AB Pav459 and further reduction in FO (1.75% FO; 20% AB). Replacing FM with AB Pav459 showed no significant effects on the growth performance of Atlantic salmon. Fish across all diets exhibited growth exceeding 200% from their initial weight. Analysis of total lipid content after the 12-week trial revealed no significant differences among the diets. However, individual proportions of omega-3 (ω3) and omega-6 (ω6) fatty acids varied. Fatty acid profiling in muscle and liver tissues showed distinct compositions reflective of dietary treatments. Linoleic acid (LA) and α-linolenic acid (ALA) exhibited higher proportions in total fatty acids than in membrane lipids. Docosahexaenoic acid (DHA) emerged as the predominant fatty acid in the membranes of both liver and muscle tissues. Furthermore, an analysis of sterol composition in Pavlova and salmon muscle tissue showed the presence of important sterols, including conventionally animal-associated cholesterol. This emphasizes the suitability of microorganisms, such as Pav459, for synthesizing diverse nutrients. Stable isotope analysis demonstrated direct incorporation of eicosapentaenoic acid (EPA) and DHA from diets into salmon tissues. Notably, minimal biosynthesis from the precursor ALA was observed, reaffirming the utility of Pav459-derived fatty acids. The EPA+DHA proportions in the fillet consistently met daily human consumption requirements across all dietary conditions, supporting the use of Pav459 algal biomass as an alternative to FM.