Hofmeister's father, a music and book publisher from Leipzig, was also a keen amateur botanist and encouraged his son's interest in botany. Wilhelm left school at 15 and served a two-year apprenticeship in a music shop before entering his father's business in 1841. He soon began to study botany seriously in his spare time and was greatly influenced by the views of Matthias Schleiden, who believed that botany could advance rapidly if researchers concentrated on studying cell structure and life histories.

Using procedures recommended by Schleiden, Hofmeister's first work was to disprove Schleiden's theory that the plant embryo develops from the tip of the pollen tube. He believed that a preexisting cell in the embryo sac gave rise to the embryo and his paper The Genesis of the Embryo in Phanerogams (1849) gained him an honorary doctorate from Rostock University.

Hofmeister's major discovery, however, was to demonstrate the alternation of generations between sporophyte and gametophyte in the lower plants. The work, published in 1851 as Vergleichende Untersuchungen (Comparative Investigations), showed the homologies between the higher seed-bearing plants (phanerogams) and the mosses and ferns (cryptogams) and demonstrated the true position of the gymnosperms between the angiosperms and the cryptogams. In 1863 Hofmeister was appointed professor at Heidelberg University and director of the botanic gardens there, and in 1872 moved to Tübingen University to succeed Hugo von Mohl.