As global demand for affordable, high-quality protein continues to rise, commercial aquaculture has emerged as a critical solution. The industry’s reliance on ocean-derived fishmeal (FM) and fish oil (FO) for aquafeed poses sustainability concerns. Marine microalgae offer a promising alternative due to their comparable nutrient profiles and potential for large-scale, sustainable production. We conducted a feeding trial with juvenile rainbow trout to evaluate fish-free, microalgae-based diets as alternatives to FM and FO. Diets were formulated using Nannochloropsis oculata (N. oculata) by-product as a protein source and Schizochytrium sp. (whole-cell or oil) as a source of DHA and key antioxidants. Diets included a reference formulation containing FMFO and three experimental diets: 75% replacement of FMFO using N. oculata and Schizochytrium whole-cell (NSW75), 100% replacement using both (NSW100), and 100% replacement using N. oculata and Schizochytrium oil (NSO100). The NSW100 diet supported growth and feed conversion comparable to the FMFO control; the NSO100 had lower growth than the FMFO control. Cost analysis revealed that fish-free diets had the numerically lowest, though not significantly different, values for formulated feed costs and economic conversion ratios, highlighting their commercial potential. These findings demonstrate that microalgae-based aquafeeds combining N. oculata and Schizochytrium sp. can viably replace FMFO.