Drone brood is a little-known bee product which is frequently considered as a male equivalent of royal jelly and is sometimes used as its adulterant. The aim of the study was to compare the chemical composition and biological activity of both bee products originated from the same apiaries (n = 3) limiting the influence of genetic and environmental factors. Moreover, for drone brood study covered testing three stages of larval development (days 7, 11, and 14). The comparison included mineral composition (ICP-OES method), protein content and protein profile (SDS-PAGE), testosterone and estradiol content (ELISA tests). HPTLC method was used to analyze of sugar, amino acids, and polyphenolic profile of drone brood and royal jelly. Moreover, their antioxidant and enzymatic properties were compared. A lot of similarities between drone brood and royal jelly were found in terms of chemical components. However, drone brood was more abundant in iron and manganese, reducing sugars and some amino acids, especially proline, tyrosine, and leucine. It contained more testosterone (especially on the 14th day) and estradiol (on the 7th day). The greatest differences in the enzymatic activities and polyphenolic profile were found. Diastase and α-glucosidase activity were found as specific enzymes of the drone brood. Similarly, ferulic and ellagic acids were characteristic for brood and were not present in royal jelly. The study showed a lot of similar features for both tested bee products, however, some specific markers which can serve to differentiate drone brood and royal jelly were found.