<p>Acne vulgaris (AV) is a persistent inflammatory skin disease that affects the pilosebaceous units. Oral azithromycin has shown significant effectiveness in treating AV, attributed to its robust anti-inflammatory and antimicrobial properties. This study aimed to evaluate the efficacy of azithromycin in patients with moderate-to-severe AV, with a focus on its influence on sebum composition and specific biomarkers, including pyridoxine (B6), biotin (B7), cobalamin (B12) (measured by HPLC), and homocysteine (measured by ELISA). A case-control study was conducted at the dermatology department of Al-Imamain-Alkadhimain Medical City, Baghdad. The study included thirty patients aged 15–35 years with moderate-to-severe AV, and thirty healthy individuals of the same age and sex served as a control group. Patients received 500&#xa0;mg of azithromycin on alternate days for 12 weeks. Laboratory and clinical parameters were assessed at baseline and compared to the control group, then re-evaluated after treatment. The findings indicated a significant decrease in serum levels of folic acid, B7, and B6 at both 6 and 12 weeks post-treatment (<i>p</i> &lt; 0.05). Serum homocysteine levels increased slightly over time, but this change was not statistically significant (<i>p</i> &gt; 0.05). No significant difference in serum B12 levels was observed at 6 weeks (<i>p</i> &gt; 0.05); however, a significant increase was noted at 12 weeks (<i>p</i> &lt; 0.05). Clinical score, skin moisture, and sebum squalene (Sebum-SQ) content improved significantly post-treatment (<i>p</i> &lt; 0.05), while sebum cholesterol (Sebum-Chol.) levels showed significant change only at 12 weeks (<i>p</i> &lt; 0.05). The finding supports the efficacy of azithromycin in managing inflammatory acne and modulating sebum composition and specific biomarkers. However, the relatively small sample size and absence of power analysis limit the generalizability of the results. Future studies with larger cohorts are recommended to validate these findings.</p>