AbstractRoyal jelly, due to its unique bioactive components, has special biological activities, but a great extent of its nutritional value is lost during processing and storage. Lyophilization, an effective preservation technique, can feasibly preserve the main bioactive compounds present in royal jelly. In this study, fresh royal jelly was subjected to the freeze‐drying process at a pressure and temperature of 100 Pa and − 70°C, respectively, for 40 h. The results obtained indicated that the pH, turbidity, total phenol content, and antioxidant activity of the royal jelly powder (RJP), during 3 months of storage at ambient temperature (30°C), were constant with values of 4.30, 1.634 (%A.U.), 0.617 (g/L), and 28.7 (%), respectively. Moisture content of the prepared RJP was less than 1%, while that of the fresh royal jelly was 70%. Furthermore, for the fresh royal jelly, the mentioned parameters were significantly (p< .05) decreased after 2 months of storage at freezer temperature (−20°C). GC–MS analysis indicated that the amount of 10‐hydroxy‐2‐decanoic acid (10H2DA) in RJP was 3.85 times more than that of fresh royal jelly. The obtained results also indicated that prepared RJP had a high bactericidal effect towardEscherichia coliandStaphylococcus aureus, with clear zone diameters of 12 and 15 mm, respectively. The present study provides a foundation for research on the potential application of prepared RJP and the development of dietary supplements and functional foods.