In the present study, the effect of four Vajikaran Rasayana herbs on penile erection, sperm count, seminal fructose content in vivo and nitric oxide (NO) release in vitro was assessed. Lyophilised aqueous extracts of Asparagus racemosus Willd. (AR), Chlorophytum borivilianum Sant. F. (CB), Curculigo orchioides Gaertn. (CO), and Dactylorhiza hatagirea (D. Don) Soo (DH) were orally administered at 100 mg/kg body weight to Wistar strain male albino rats. Penile erection index and sperm count were determined by visual observation; the seminal fructose concentration was measured spectrophotometrically using resorcinol reagent; and NO release was assessed in a mouse macrophage cell line (RAW264) spectrophotometrically using a commercial Griess reagent kit. Penile erection index, sperm count, seminal fructose concentration and in vitro NO release were the parameters measured. A significant effect on the sperm count, seminal fructose content and penile erection index was observed upon treatment with the extracts. The effect of extracts on inducible NO release in vitro directly correlated with the enhanced erectile function in vivo. The aphrodisiac claims attributed to the four Vajikaran Rasayana herbs were tested and a distinctive effect of all extracts tested was observed, with C. borivilianum showing a highly significant response for all parameters measured in vivo and in vitro. The present study also provides a good correlation between the in vivo improvement of penile erection and in vitro NO releasing activity of the extracts. Increase in seminal fructose levels and sperm count further validates the role of these herbs in improving reproductive function.