Born into a peasant family in Carlsbad, Bohemia, Loschmidt was educated by the local clergy, who encouraged him to pursue his studies at the Prague Gymnasium and at the German University in Prague. After graduating in 1843 he struggled with various businesses before ending his commercial career in 1854 as a bankrupt. He turned to academic life and in 1856 was appointed to the Vienna Realschule where he remained until 1868 when he moved to the University of Vienna as professor of physical chemistry.

In 1861 Loschmidt published a brief work, Chemische Studien (Chemical Studies), in which he listed 368 formulae. Like most chemists of the time, Loschmidt was seeking for ways to express chemical structure and composition accurately and graphically. In his system, atoms were represented by circles, with a large circle for carbon and a smaller one for hydrogen. Thus four years before Kekulé announced his own results, Loschmidt represented the benzene molecule by a single large ring (the carbon) with six smaller circles (hydrogen) around the rim. Little attention seems to have been paid to his work at the time.

He is far better known for a paper published in 1865 entitled Zur Grosse der Loftmolecule (On the Magnitude of Air Molecules) in which he made the first accurate estimate of the size of molecules. Using the kinetic theory of gases, Loschmidt derived the equation s = 8el, where s is the molecular diameter, l is the mean free path (the distance a molecule moves between successive collisions), and e is the condensation coefficient. This latter factor was derived from changes in volume due to evaporation and condensation. Using published data for e and l, he obtained the value 10^–7 centimeter. In his honor, the number of particles per unit volume of an ideal gas at standard temperature and pressure is known as Loschmidt's constant (or Loschmidt's number). It has the value 2.686763 × 10²⁵ m^–3.