In 1832, lacking his own laboratory facilities at Kassel, Wöhler worked with Justus Liebig in his Giessen laboratory. In that year, Wöhler and Liebig published an investigation of the oil of bitter almonds. Through their detailed analysis of the chemical composition of this oil, they proved by their experiments that a group of carbon, hydrogen, and oxygen atoms can behave chemically as if it were the equivalent of a single atom, take the place of an atom in a chemical compound, and be exchanged for other atoms in chemical compounds. Specifically, their research on the oil of bitter almonds showed that a group of elements with the chemical composition CHO can be thought of as a single functional group, which came to be known as a benzoyl radical. In this way, the investigations of Wöhler and Liebig established a new concept in organic chemistry referred to as compound radicals, which had a profound influence on the development of organic chemistry. Many more functional groups were later identified by subsequent investigators with wide utility in chemistry.
Liebig and Wöhler explored the concept of chemical isomerism, the idea that two chemical compounds with identical chemical compositions could be different substances because of different arrangements of the atoms in the chemical structure. Aspects of chemical isomerism originated in the research of Berzelius. Liebig and Wöhler investigated silver fulminate and silver cyanate. These two compounds have the same chemical composition yet are chemically different. Silver fulminate is explosive, while silver cyanate is a stable compound. Liebig and Wöhler recognized these as examples of structural isomerism, which was a significant advance in understanding chemical isomerism.
Wöhler has also been regarded as a pioneering researcher in organic chemistry as a result of his 1828 demonstration of the laboratory synthesis of urea from ammonium cyanate, in a chemical reaction that came to be known as the "Wöhler synthesis". Urea and ammonium cyanate are further examples of structural isomers of chemical compounds. Heating ammonium cyanate converts it into urea, which is its isomer. In a letter to Swedish chemist Jöns Jacob Berzelius the same year, he wrote, In a manner of speaking, I can no longer hold my chemical water. I must tell you that I can make urea without the use of kidneys of any animal, be it man or dog.
Wöhler's demonstration of urea synthesis has become regarded as a refutation of vitalism, the hypothesis that living things are alive because of some special "vital force".
It was the beginning of the end for one popular vitalist hypothesis, the idea that "organic" compounds could be made only by living things.
In responding to Wöhler, Jöns Jakob Berzelius  acknowledged that Wöhlers results were highly significant for the understanding of organic chemistry, calling the findings a "jewel" for Wöhlers "laurel wreath". Both scientists also recognized the work's importance to the study of isomerism, a new area of research.
Wöhlers role in overturning vitalism is said to have become exaggerated over time. This tendency can be traced back to Hermann Kopps History of Chemistry (in four volumes, 1843–1847). He emphasized the importance of Wöhler's research as a refutation of vitalism but ignored its importance in understanding chemical isomerism, setting a tone for subsequent writers.
The notion that Wöhler single-handedly overturned vitalism also gained popularity after it appeared in a popular history of chemistry published in 1931, which, "ignoring all pretense of historical accuracy, turned Wöhler into a crusader".
Contrary to what was thought in Wöhler's time, cyanate is not a purely inorganic anion, as it is formed in various metabolic pathways. Thus the conversion of ammonium cyanate into urea was not an example of production of an organic compound from an inorganic precursor.