Honey, valued for its nutritional and antimicrobial benefits, has experienced an increased production in recent decades. However, this rise has been accompanied by concerns of adulteration, often involving the fraudulent addition of sugars. Our study sought to compare the physicochemical and isotopic properties of various honeys available to Chilean consumers, assessing the extent of adulteration. Samples included honey produced from bees that fed on multiple flowers and those fed by ulmo flowers – an endemic species of South America that produces a high-quality, high-cost honey – and analyzed for antimicrobial activity against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. The analysis of ash content (%), moisture (%), pH and total phenolic content (mg GAE/100 g honey) found little obvious differences among honeys regardless of the feeding flowers (multifloral vs ulmo), type of purchase market (formal vs informal) or origin of the honeys (Central vs Southern Chile). However, the use of stable isotope analysis (δ13C and δ15N) of honey provided a powerful means to identify the degree of adulteration prior to the point of sale. Multifloral honeys purchased at informal markets were all adulterated, and ulmo honeys included both the least and most adulterated honeys. Regarding their antimicrobial activity, most multifloral honeys were less effective than ulmo honeys. Notably, while multifloral honey activity was independent of adulteration, the antimicrobial activity of ulmo honey was negatively affected by adulteration. Display omitted •Significant lower δ13C values (pointing to adulteration) were observed in multifloral honey.•Honeys purchased at formal markets were unadulterated.•Ulmo honeys were the least and most adulterated honeys, associated to market type.•Most of the ulmo honeys had a higher antimicrobial activity than multifloral honeys.•Independently of adulteration, all multifloral honeys had the same antimicrobial activity.