Apis mellifera beeswax is synthesized from honey sugars and secreted by specialized glands located on the ventral side of the abdomen of worker bees aged 12–18 days. This beeswax possesses various therapeutic properties beneficial to human health. Given the priority in pharmacological research to develop new drugs from natural sources with minimal side effects, the absence of studies on the biological activities of A. mellifera beeswax is notable. Therefore, this study aims to investigate the chemical composition, as well as the anti-inflammatory, antiviral, cytotoxic, antimicrobial, and antioxidant activities of hydroethanolic crude beeswax extract from A. mellifera. The results revealed the presence of the flavonoids rutin (19.39 µg/g), naringenin (69.52 µg/g), daidzein (3.34 µg/g), quercetin (53.69 µg/g), kaempferol (51.88 µg/g), hesperetin (8.13 µg/g) and the phenolic gallic acid (421.13 µg/g), chlorogenic acid (878.80 µg/g), methyl gallate (56.21 µg/g), caffeic acid (8.83 µg/g), syringic acid (8.07 µg/g), coumaric acid (9.80 µg/g) and cinnamic acid (4.94 µg/g) which assessed by HPLC. Apis mellifera beeswax had considerable anti-inflammatory activity percent hemolysis Inhibition was 90.2% at concentration 1000 µg/mL, while indomethacin (standard drug) showed 100% activity at the same concentration. Also, exhibited major antioxidant activity as assessed by DPPH radical scavenging activity assay which compared to ascorbic acid, the IC50 of A. mellifera beeswax was 16.93 µg/mL. The antiviral activity of A. mellifera beeswax against HAV was recorded at 78.52%, while it was 24.2% against CoxB4 at the same concentration. The beeswax of A. mellifera demonstrated greater inhibitory activity than the reference compound gentamicin against Staphylococcus aureus (25 ± 0.18 mm) and Escherichia coli (15 ± 0.81 mm). It also showed significant inhibitory activity against Candida auris (22 ± 0.21 mm). Moderate inhibitory effects were observed against Klebsiella pneumoniae (19 ± 0.36 mm), Enterobacter aerogenes (14 ± 0.46 mm), and Candida albicans (15 ± 0.13 mm). The minimum lethal concentrations and minimum inhibitory concentrations of A. mellifera beeswax against these pathogenic strains ranged from 125 to 250 μg/mL and 62.5–125 μg/mL, respectively. The findings of this study highlight the substantial biological potential of beeswax extract, suggesting that A. mellifera beeswax warrants further investigation as a source for new pharmacological alternatives. However, additional in vitro and in vivo studies are necessary to fully assess its potential benefits for human health.