The aim of this study was to investigate the chemical composition of powdered mealworm larvae (Tenebrio molitor L.) reared on different nutritional substrates. Wheat bran was used as the control substrate, while, barley (whole grain), oats (whole grain), oats and barley whole grain mixture (50:50), buckwheat, and mixture of oat and barley sprouts (50:50) were selected as experimental substrates. A proximate analysis and mineral content determination were carried out for all substrates and larvae. Since insects are becoming an attractive alternative protein source for poultry, pigs and fish as a “novel” and natural feed material, lysine, methionine, and threonine levels have also been determined. Furthermore, in addition to fat content, the fatty acid profile was also determined. It was found that wheat bran was the most suitable substrate in terms of high protein yield in larvae (71% dry weight) with lowest fat content (7% dry weight). Linoleic acid content was the highest in the larvae fed wheat bran, while the highest α-linolenic acid content was obtained in the larvae reared on a mixture of oat and barley sprouts (50:50). Moreover, linear regression analysis demonstrated a weak correlation of substrate and larval protein content for all selected substrates. The highest content of each mineral was also obtained in the larvae reared on wheat bran (except iron and manganese, which were the second highest). Based on the experimental results, it can be concluded that meals from T. molitor larvae are an excellent feed material for use in livestock diets, especially those reared on wheat bran.