<p>Cobalt stress can promote the accumulation of dendrobine-type total alkaloids (DTTAs) in <i>Trichoderma longibrachiatum</i> UN32; however, the signalling mechanisms underlying this response remain unclear. In this study, we examined whether changes in Ca²⁺ and nitric oxide (NO) signalling were associated with ROS-related responses and cobalt-enhanced DTTAs production under cobalt stress. Co²⁺ treatment increased DTTAs accumulation together with intracellular hydrogen peroxide (H₂O₂), Ca²⁺ and NO levels. Among Ca²⁺ signalling inhibitors, neomycin (Neo) showed the strongest effect: 6 mM Neo reduced intracellular Ca²⁺ by 59.86%, DTTAs production by 33.70% and H₂O₂ levels by 25.50% compared with CoCl₂ treatment. Time-course analysis further showed coordinated changes in H₂O₂, Ca²⁺ and DTTAs accumulation during fermentation. In contrast, L-NAME-mediated inhibition of NO synthesis did not significantly affect DTTAs production. These findings suggest that ROS-Ca²⁺ interplay is associated with cobalt-enhanced DTTAs accumulation in <i>T. longibrachiatum</i> UN32, whereas NO has a limited role under the tested conditions.</p>