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In this work, for the first time, a gruyt beer and the same one after the addition of Citrus aurantium essential oil (AEO), were investigated to determine the composition of the volatile fraction. The applied analytical techniques, such as Head Space/Solid Phase Microextraction-Gas Chromatography-Mass Spectrometry (HS/SPME-GC-MS) and Proton Transfer Reaction-Time of Flight-Mass Spectrometer (PTR-ToF-MS), allowed us to identify the content of volatile organic compounds (VOCs). From the comparison between the two beer samples, it showed that the one after the addition of AEO was particularly richened in limonene and a series of minor terpene compounds. AEO was also characterized by GC/MS analysis and the results showed that limonene reached 95%. Confocal microscopy was used to look at riboflavin autofluorescence in yeast cells. It was found that beer with AEO had twice as much fluorescence intensity as the control. A spectrophotometric analysis of total polyphenols, tannins, and flavonoids, and a bioactivity screening, including 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-Azinobis-(3-Ethylbenzthiazolin-6-Sulfonic Acid) (ABTS) radical scavenger, chelating, reducing, antiglycative ones, were also carried out. Moreover, the tolerability of the tested samples in human H69 cholangiocytes and the cytoprotection towards the tert-butyl hydroperoxide (tBOOH)-induced oxidative damage were evaluated. Under our experimental conditions, the beers were found to be able to scavenge DPPH and ABTS radicals and chelate iron ions, despite weak antiglycative and reducing properties. The tested samples did not affect the viability of H69 cholangiocytes up to the highest concentrations; moreover, no signs of cytoprotection against the damage induced by tBOOH were highlighted. Adding AEO to beer resulted in a moderate enhancement of its DPPH scavenging and chelating abilities, without improvements in the other assays. Conversely, AEO and its major compound limonene were ineffective when assessed at the concentrations added to beer. This evidence suggests that the addition of AEO may enhance the organoleptic features of the beer and slightly potentiate some of its bioactivities. [ABSTRACT FROM AUTHOR]