The multifunctional roles of melatonin (ML) and hydrogen sulfide (H2S) as plant gasotransmitters are well understood. However, their interaction in defensive responses is still unclear. Here, we assessed how the ML-H2S interaction impacts tomato plants exposed to arsenic (As, 10 mg/L) toxicity. The results showed that ML and ML+H2S treatments alleviated As toxicity by inducing the transcription of ANS, PAL, DFR, CHS, F3H, and CHI and an enhanced accumulation of phenolic and anthocyanin compounds. ML and ML+H2S led to a drop in the transcription of PT2 and Lsi1, resulting in reduced As uptake and subsequent movement to the leaves. ML and ML+H2S treatments resulted in high GSH1, PCS, and ABC1 transcription. This led to elevated levels of glutathione and phytochelatins, facilitating As sequestration in the roots and protecting root and leaf cells against As. ML and ML+H2S treatments enhanced tomato growth under As treatments by optimizing chlorophyll metabolism, increasing internal H2S, and amplifying the antioxidant machinery function. In contrast to the sole use of ML treatment, ML+H2S exhibited more pronounced positive impacts on As-stressed tomatoes, indicating synergistic/interactive effects between these two substances. On the other hand, the use of hypotaurine, an H2S scavenger, demonstrated the inhibition of the beneficial impact of ML and ML+H2S treatments in stimulating defensive responses, which highlights the substantial involvement of H2S in the signaling pathway activated by ML during As stress. Hence, we can conclude that ML requires the presence of endogenous H2S to mitigate the adverse effects of As on tomato seedlings.