Main conclusion <p>The study demonstrates that <i>Macrocoma orthotrichoides</i> employs a poikilochlorophyllous strategy and exhibits rapid photosynthetic recovery, providing novel biochemical and fluorescence-based evidence of desiccation tolerance in this species.</p> Conclusion <p>Mosses are poikilohydric plants that activate defence mechanisms to protect their tissues and metabolism from dehydration-induced damage, particularly by counteracting the generation/accumulation of reactive oxygen species (ROS). In this study, we evaluate the responses of <i>Macrocoma orthotrichoides</i> gametophytes to dehydration by analysing chlorophyll <i>a</i> fluorescence parameters, metabolite accumulation, and antioxidant enzyme activities in response to ROS production under varying humidity conditions. Fresh gametophyte samples were exposed to controlled moisture regimes, and biochemical analyses revealed that the activity of antioxidant enzymes and proline levels fluctuated in response to dehydration; however, these changes did not fully mitigate oxidative stress and ROS accumulation. Changes in photosynthetic pigment concentrations mirrored enzymatic activity, being consistent with humidity conditions. The decrease in chlorophyll and carotenoid levels during desiccation indicates a poikilochlorophyllous strategy in <i>M. orthotrichoides</i>, with pigments and thylakoid structures being restored upon rehydration. Fluorescence analysis demonstrated that this species tolerates intense dehydration and rapidly regains photosynthetic capacity upon rehydration. Overall, our findings indicate that <i>M. orthotrichoides</i> possesses a suite of biochemical, enzymatic, and physiological adaptations that enable survival and recovery in fluctuating moisture environments, thereby advancing our understanding of desiccation tolerance and photosynthetic resilience in mosses.</p> Graphical abstract <p><i>Macrocoma orthotrichoides</i> shows desiccation tolerance and photosynthetic recovery after rehydration.</p> <p></p>