Bioactive amines (BA) have important neurological and antioxidant functions in the human body, in addition to acting in the prevention and reduction of symptoms caused by COVID-19. This study aimed to evaluate those metabolites and their precursors amino acids in honeys collected in Santa Catarina and São Paulo states (Brazil). Analyzes were performed by HPLC and data were submitted to multivariate analysis. Tryptophan (Trp), L-dopa (L-Dop), and tryptamine (Tryp) stood out for their contents in SC-honeys from agroecological zones of altitude as Campos de Lages (Trp: 9483.94, L-Dop: 0.46, and Tryp: 197.41 µg/100 g), Vale do Rio do Peixe (Trp: 7797.87, L-Dop: 0.93, and Tryp 191.98 µg/100 g), and Planalto Serrano São Joaquim (Trp: 7337.70, L-Dop: 0.91, and Tryp: 159.52 µg/100 g). In contrast, dopamine, serotonin, and melatonin were detected in coastal and valley regions (Cfa). Honeys from the region of Vale do Itajaí, with a predominant cinnamon flowering, presented higher levels of dopamine (39.61 µg/100 g), as those from Litoral de Laguna and Vale do Itajaí showed higher levels of serotonin and melatonin (748.38 and 127.01 µg/100 g, respectively). For the SP honeys, appreciable amounts of Trp (1158.59 µg/100 g) were detected in samples from Avaré (Cfa). Results reveal that the amino acids and BA levels in honeys vary depending on the region of production, as well as on the nectar donor plants. This analysis, in addition to being important for the identification of compounds with pharmacological activities, can also be used as biochemical markers identification of honey’s botanical origin. This is a pioneering study regarding the investigation of BA in honeys from different ecological zones of Brazil. The search for substances with important biological properties creates new opportunities, such as the emergence of market niches for honey, stimulating the beekeeping activity, especially in the context of family farming.