The understanding of stomatal regulation in climate stress is essential for ensuring resilient crops. The investigation of the stomatal regulation in combined heat and drought stress aimed to link effects of exogenous melatonin on stomatal conductance (g _s) and its mechanistic interactions with ABA or ROS signaling. Melatonin-treated and non-treated tomato seedlings were subjected to moderate and severe levels of heat (38°C for one or three days) and drought stress (soil relative water content of 50% or 20%) applied individually and in combination. We measured g _s, stomatal anatomy, ABA metabolites and enzymatic ROS scavengers. The stomata in combined stress responded predominantly to heat at soil relative water content (SRWC) = 50% and to drought stress at SRWC = 20%. Drought stress increased ABA levels at severe stress, whereas heat stress caused an accumulation of the conjugated form, ABA glucose ester, at both moderate and severe stress. The melatonin treatment affected g _s and the activity of ROS scavenging enzymes but had no effect on ABA levels. The ABA metabolism and conjugation of ABA might play a role in stomatal opening toward high temperatures. We provide evidence that melatonin increases g _s in combined heat and drought stress, but the effect is not mediated through ABA signaling.