Topologically protected magnetic skyrmions are expected to be used in the next-generation spintronic devices. Realizing their nucleation and manipulation at room temperature is fundamental for future practical applications. Here, using in situ Lorentz transmission electron microscopy and micromagnetic simulation, we demonstrate that magnetic biskyrmions can spontaneously exist at room temperature in Nd1–xTbxCo5 (x = 0.3, 0.5) alloys. The spontaneous biskyrmions are controllably obtained over a wide temperature range across room temperature by switching atomic chemical environment. Furthermore, the density of biskyrmions can be tuned by a small magnetic field. High-density biskyrmions are stimulated to form in the thinner region at room temperature by introducing an in-plane magnetic field component. These results provide valuable insights into the manipulation of topological states, which is of great significance to their practical applications.