Kolbe was born in Elliehausen near Hanover, Germany.
He became an assistant to Robert Wilhelm Bunsen at the University of Marburg in 1842, after studying chemistry with Friedrich Wöhler. Subsequently he assisted Lyon Playfair at the University of London and from 1847 to 1851 was engaged in editing the Handwörterbuch der reinen und angewandten Chemie (Dictionary of Pure and Applied Chemistry) written by Justus von Liebig and Wöhler. Kolbe then succeeded Bunsen at Marburg, and in 1865 he went to the University of Leipzig.
At that time, it was believed that organic and inorganic compounds are independent from each other, and that organic compounds could be created only by living organisms. Kolbe believed that organic compounds could be derived from inorganic ones, directly or indirectly, by substitution processes. He validated his theory by converting carbon disulfide, in several steps, to acetic acid (1843-45). Introducing a modified idea of structural radicals, he contributed to the establishment of structural theory. He also predicted the existence of secondary and tertiary alcohols.
He worked on the electrolysis of the salts of fatty and other acids and prepared salicylic acid, a building block of aspirin. The process was named Kolbe synthesis (or Kolbe-Schmitt reaction), which works by heating sodium phenolate (the sodium salt of phenol) with carbon dioxide under pressure (100 atm, 125°C), then treating it with sulfuric acid.
With Sir Edward Frankland he found that nitriles can be hydrolyzed to the corresponding acids. As editor of the Journal für praktische Chemie (Journal of practical chemistry, 1869), he was sometimes severely critical of the work of others.