In honey, the content of proteins, including the enzymes, is relatively low and has a minor nutritive significance. On the other hand, the proteins, including the enzymes, are usually used as honey quality evaluation parameters. This is because protein content and enzyme activities vary regarding the botanical origin of the honey. Since the results of protein content, glucose-oxidase, and acid phosphatase, for honeys produced in Croatia, are not available, four of the most abundant honey types produced in Croatia (black locust, sage, chestnut, and honeydew honey) are characterised according to the protein and proline content and enzyme activities. The characterisation was done to determine specificities and contribute to the characterisation of unifloral honeys. Dark honey types (honeydew and chestnut honey) had a higher proline content, and diastase, invertase, and glucose-oxidase activity than lighter sage and black locust honey. Black locust honey has a naturally low enzyme activity and showed the highest acid phosphatase activity among the analysed honey types, while honeydew honey, otherwise known to possess high proline content and enzyme activity, had a low protein content comparable to black locust honey. Statistically significant correlations were obtained between all analysed parameters, with the exception of acid phosphatase activity.