This study investigates the potential of utilizing three food wastes: cheese whey (CW), beet molasses (BM), and corn steep liquor (CSL) as alternative nutrient sources for the cultivation of the diatom Phaeodactylum tricornutum, a promising source of polyunsaturated eicosapentaenoic acid (EPA) and the carotenoid fucoxanthin. The CW media tested did not significantly impact the growth rate of P. tricornutum; however, CW hydrolysate significantly enhances cell growth. BM in cultivation medium enhances biomass production and fucoxanthin yield. The optimization of the new food waste medium was conducted through the application of a response surface methodology (RSM) using hydrolyzed CW, BM, and CSL as factors. The results showed a significant positive impact of these factors (p < 0.005), with an optimized biomass yield of 2.35 g L-1 and a fucoxanthin yield of 3.64 mg L-1 using a medium composed of 33 mL L-1 of CW, 2.3 g L-1 of BM, and 2.24 g L-1 of CSL. The experimental results reported in this study showed that some food by-products from a biorefinery perspective could be utilized for the efficient production of fucoxanthin and other high-added-value products such as eicosapentaenoic acid (EPA).