Ocimum tenuiflorum is a sacred medicinal plant bestowed with multiple health benefits. This plant is traditionally considered an adaptogen. Many scientific studies have indicated the anti-stress potential of Ocimum tenuiflorum but with higher doses. The present study investigated the effects of HolixerTM (a clinically studied standardized Ocimum tenuiflorum extract) on modulating stress using two in vivo models, namely the swim endurance study in mice and forced swim test in rats. In addition, we explored the mechanism of action of HolixerTM on the HPA axis using two in vitro cell-based assays to check for its inhibitory effect on cortisol release and CRF1 receptor antagonistic activity. Ocimum tenuiflorum extract enhanced the swimming time in mice, reduced the stress-induced increase in immobility time, and prevented the increase in corticosterone in rats subjected to the forced swim test. Further, Ocimum tenuiflorum extract inhibited cortisol release and exhibited a significant CRF1 receptor antagonist activity. Thus, Ocimum tenuiflorum extract was found effective in managing stress, and the effect could be due to the inhibition of cortisol release and the antagonistic effect on the CRF1 receptors.