Holmberg was one of Knut Lundmark's students at the Lund observatory. In his dissertation, "A study of double and multiple galaxies" (1937) he showed that galaxies often appear in groups and pairs and he also realized that it would be possible, using statistics, to determine the masses of pairs of galaxies knowing the radial velocities of the components and this method has been very important in extragalactic research. The observations also resulted in the discovery that colours of paired galaxies are closely correlated, the "Holmberg effect".
During his studies of the origin and evolution of galaxy pairs he published, in 1941, perhaps his most original work on the effects of interacting galaxies. His experiment consisted of light bulbs representing the stellar distribution in a galaxy and the gravity simulated by the intensity of the light. The "gravitational" forces were then measured by photocells and he could, step by step, follow the event of two galaxies passing close to each other. One of the conclusions was that the galaxies often merge into one single larger galaxy and it was not until about 30 years later, when appropriate computers became available, that his results could be confirmed.
Holmberg was a skilful observer and began, in the late 1940's, developing modern methods for measuring galaxy magnitudes from photographic plates. The often used standard measure "Holmberg radius" (the radius of a galaxy out to an isophote, representing a blue magnitude of 26.5 per square arc second) is a result from this period. His work was published in a large galaxy catalogue and this catalogue has been widely used in extragalactic research, for example in many statistical studies of the space density of different types of galaxies ("Luminosity function"). Holmberg found a correlation between galaxy magnitudes and colours ("Holmberg relation") and showed that elliptical galaxies were older than spiral galaxies.
From the 1940's to the the end of the 1960's he often carried out research and lectured at the Mount Wilson and Mount Palomar observatories.
The IAU Symposium no. 44 "External Galaxies and Quasi-stellar objects" (1970) was arranged by Holmberg in Uppsala. This meeting
greatly stimulated the interest in extragalactic astronomy in Sweden.