The physicochemical properties of honey vary considerably depending on the nectar sipped by Apis mellifera bees because these characteristics are determined by environmental, geographical, and vegetational conditions in foraging areas. The aim of this study was to characterize types of honey from the Biobío Province in the Biobío Region, Chile, according to its botanical origin and physicochemical and microbiological characteristics, and thus contribute to increased knowledge about honey in the region. A total of 11 samples from different communes within the region were analyzed. Physicochemical parameters such as moisture, insoluble solids, ashes, electrical conductivity, pH, and hydroxymethylfurfural were determined, and the presence of coliforms and Escherichia coli was evaluated. Two places of provenance were characterized as monofloral of introduced plant species: the M11 sample of Melilotus indicus and the M1 sample of Echium vulgare. One place of provenance (M6 sample) was characterized as monofloral of Eucryphia glutinosa, species endemic to Chile. All types of honey complied with the physicochemical standards established by national and international food safety regulations, and none had any signs of coliforms or E. coli. Results showed that there are new types of honey in the region that meet all the required parameters; in addition, they provide a comparison of plant species that are unique in the world. This demonstrates the potential of these types of honey for national and international marketing.