Genital ulcer disease is most commonly caused by types I and II of the herpes simplex virus (HSV). These viruses also cause infections of the genitalia. To evaluate the prevalence of HSV infection in Egypt and its treatment with hesperidin extract, this study was conducted. The antiviral and cytotoxic properties of hesperidin were thus assessed on 200 blood samples that were obtained for different types of analysis. Twenty (10%) of the 200 samples tested positive for HSV-I, and seven (3.5%) tested positive for HSV-II, according to our results. Furthermore, 6.75 log (10)/0.1 mL and 7.24 log (10)/0.1 mL, respectively, were the infectivity titers of HSV-I and HSV-II. HSV-I had overall values by PCR of 401–904 with a mean of 665.7 ± 163.5, while the HSV-II had overall values of 270–900 with a mean of 598.6 ± 243.4. There was no statistically significant difference between the HSV-I and HSV-II results by PCR, according to a statistical study between the two viruses (P = 0.517). Hesperidin's antiviral activity was seen to be 11% and 13.8% inhibition against HSV-I and HSV-II viruses, respectively, at a safe dosage of 312. 5 ug/mL and an IC₅₀ of 682.22 ug/mL. Pre- and post-treatment analyses revealed hesperidin's mode of action, which was more effective against HSV-II than HSV-I (as revealed by the post-treatment approach). Finally, hesperidin's IC₅₀ against HSV-II was 682.22 ug/mL, indicating a stronger viricidal impact.