Propolis is derived from plant resin and bees' salivary secretions, are commonly used in folk medicine. Studies on propolis from stingless bees have increased in recent years, highlighting their relevance due to the variety of species and the bioactive components. However, scientific records confirming these activities in species occurring in Brazil are still scarce. In that context, this study aims to determine the chemical profile and the antioxidant, antifungal, and cytotoxic activities of the stingless bees Tetragona clavipes propolis. The hydroalcoholic extract was prepared, and its chemical profile was determined by Fourier transform ion cyclotron resonance mass spectrometry combined with a direct infusion electrospray ionization. Total polyphenols and flavonoid content were determined by colorimetric methods. We determined the antioxidant activity by the ability to scavenge the free radicals 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). The antifungal activity was evaluated by determining the minimum inhibitory concentration and minimum fungicidal concentration against Aspergillus fumigatus, Trichophyton rubrum, Candida albicans, and Candida parapisilosis. The results revealed high contents of total phenolics and promising antioxidant and antifungal activities. T. clavipes propolis from the rainy season showed better antioxidant and antifungal activities and higher content of flavonoids, with less variability of chemical species. The extracts exhibited fungistatic activity for C. albicans, C. parapisilosis, and A. fumigatus and fungicide for T. rubrum. Additionally, a sample collected during the rainy season demonstrated synergism with fluconazole and amphotericin B for T. rubrum and additivity for C. albicans and C. parapisilosis. In summary, the results of T. clavipes propolis revealed elevated levels of total phenolics, indicating its potential as a rich source of bioactive compounds, significant antioxidant activity, and promising antifungal activity against common pathogenic fungi. These findings contribute to our understanding of the therapeutic potential of T. clavipes propolis and provide a basis for further research and development of natural products for various applications in medicine and healthcare.