Karl Sebastian von Pfeufer (22 December 1806 – 13 September 1869) was a German physician who was a native of Bamberg.

He studied at Erlangen and Würzburg, and afterwards served as an assistant to Johann Lukas Schönlein (1793–1864).

In 1852 he was named clinical professor in the second medical division at the general hospital in Munich.

Karl Pfeufer is remembered for his collaboration with anatomist Friedrich Gustav Jacob Henle (1809–1885), an association that began in the early 1840s at Zurich.

The two doctors are credited as forerunners of German scientific medicine, and were pioneers in their attempts to create a synthesis between laboratory and clinic.