Despite the recent approval of stingless bee honey to the Argentine Food Code, there are still many gaps in information. Likely, the main reason for this is that multiple ecological and chemical factors influence their production and antimicrobial properties. This work combined metabolomic, microbiological, and physicochemical analyses to characterize the honey ofTetragonisca fiebrigifrom Northeastern Argentina. The antimicrobial activity tests showed that honey samples (n = 24) inhibited some Gram-positive and Gram-negative bacteria at different sensitivity levels. Furthermore, samples selected for their high bioactivity revealed crystallizations, a positive correlation with fungal growth, and the presence of flavonoids. The major polyphenols annotated by liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis and supported by metabolomic tools were quercetin 3,4′-dimethyl ether, pachypodol, jaceoside, irigenin trimethyl ether, corymboside, chrysoeriol 7-neohesperidoside, and corymboside. In contrast, samples missing antimicrobial activity did not crystallize, lacked flavonoids, and were enriched in phenylethylamides. Based on these findings, we discuss the significance of flavonoids and phenylethylamides on honey’s antimicrobial activity and food quality and how they may indeed reflect essential parameters of the hive, such as microbial balance and eubiosis.