Despite the challenging conditions in the pre-Saharan areas of Algeria, such as weak plant cover and a harsh climate, beekeeping is being developed and spread. In the present work, honey samples collected from ten locations in the El Oued region were examined during the spring of 2021. A melissopalynological analysis was carried out, followed by a floristic investigation. The 10 honey samples were also investigated for their physicochemical properties and antioxidant and antibacterial activity against five strains: Escherichia coli, Staphylococcus aureus, Bacillus subtilus, Listeria innocua, and Micrococcus luteus. The floristic analysis found 65 species belonging to 33 botanical families, with a dominance of the Asteraceae family accounting for 18.461% of the total. The melissopalynological study revealed only one monofloral honey (Ziziphus lotus), whereas the nine others were multi-floral. The honey’s color changed from light to dark amber, and most tested honey was of high quality, fulfilling international criteria. The total phenol and flavonoid contents varied considerably amongst the various honey samples. Furthermore, LC-MS-MS phenolic profile analysis identified the presence of 20 chemicals, of which only three phenols were found in all honey types. Antioxidant capacity analyzed with FRAP test and antiradical activities against DPPH differed from one honey sample to another. Moreover, a significant correlation was recorded between the antioxidant activity, honey’s color, polyphenol, and flavonoid contents. The S. aureus strain was the most sensitive regarding honey antibacterial activity, while M. luteus and B. subtilis strains were only moderately sensitive.