Mealworms provide a viable option for transforming agricultural and food processing by-products that can be converted into a valuable source of high-quality protein and fat suitable for both animals and humans. Hence, our investigation was aimed at employing sprouted and green potatoes, wheat bran, and by-products from brewers’ production as a comprehensive substrate for rearing mealworms. The nutritional value (fat and protein content) and composition of amino acids (AAs) and fatty acids (FAs) were tested in lyophilized and milled larvae. The results showed that the highest amount of protein was detected in sample 3L (59.18 ± 007%), grown on brewers’ spent grain, whereas sample 2S (with wheat bran) comes out with the highest fat content (34.22 ± 0.491%). It was found that the amount of FAs in the larvae depended on the substrate used: the statistically highest amount of monounsaturated FAs was detected in the sample with sprouted potatoes (1L), the highest content of omega-3 FAs was found in the control sample (4CL), with agar–agar gels, and the sample with brewers spent grains (3L) showed a statistically higher amount of oleic acid. Meanwhile, the highest content of total polyunsaturated FAs (36.23%) was detected in the sample with wheat bran (2L). During the study, 16 distinct AAs have been analyzed, and sample 3L has demonstrated the highest content in 11 instances. It is noteworthy that the sample containing brewers’ spent grain exhibited the highest peroxide levels, indicating oxidation. Meanwhile, the determined number of volatile fatty acids did not exceed the regulatory limits for meat. In summary, it can be asserted that adjusting the substrate is essential for extracting specific amino acids and FAs. However, this could potentially affect the content of peroxides. This adaptation enables the desired chemical composition in larvae, making it suitable for specific purposes such as animal or human nutrition and health enhancement.