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Rearing insects on agro-industrial by-products is a sustainable strategy for the circular economy while producing valuable products for feed and foods. In this context, this study investigated the impact of larvae diet containing agro-industrial by-products on the contents of fatty acids and sterols of <i>Ephestia kuehniella</i> (Zeller) (Lepidoptera: Pyralidae), <i>Tenebrio molitor</i> (L.) (Coleoptera: Tenebrionidae), and <i>Hermetia illucens</i> (L.) (Diptera: Stratiomyidae). For each insect, selected diets were formulated using single or combined agro-industrial by-products (i.e., apricot, brewer’s spent grain and yeast, and feed mill) and compared to a control diet. Fatty acid profiles showed differences depending on diet composition, but mostly depended on species: <i>H. illucens</i> was characterized by the abundance of C12:0, C16:0 and C18:2, whereas C:16, C18:1(n-9c), and C18:2(n-6c) were predominant in <i>T. molitor</i> and <i>E. kuehniella</i>. Sterols significantly varied as a function of diet composition and species. <i>H. illucens</i> showed low cholesterol levels and high campesterol and β sitosterol levels (0.031, 0.554 and 1.035 mg/g, respectively), whereas <i>T. molitor</i> and <i>E. kuehniella</i> had high cholesterol and low campesterol contents (1.037 and 0.078 g/kg, respectively, for <i>T. molitor</i>; 0.873 and 0.132 g/kg, respectively, for <i>E. kuehniella</i>).