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The sustainable production of freshwater from seawater desalination is receiving increasing attention. Recently, some desalination technologies are taking advantage from the coupling with renewable resources; among them, membrane distillation (MD) is one of the most promising since it can be easily powered by low-grade thermal energy. MD being an emerging technology, efforts are required to optimize geometry and operating conditions of real units in order to reduce the unitary freshwater production cost. In particular, temperature polarization is a well-known detrimental effect for the process driving force; spacers are traditionally used to enhance mixing and make temperature boundary layers thinner, at the cost of higher pressure losses. The present work is devoted to testing and comparing the performance of two different two-layer net spacers: overlapped and woven. Investigations were carried out both by experiments and by computational fluid dynamics (CFD) at different Reynolds numbers, ranging from creeping flow to turbulent flow regimes. Experiments (for intermediate to high Re) made use of thermochromic liquid crystals along with digital image processing. Computational results (for low to intermediate Re) were obtained via steady state (low Re) or direct numerical simulations (intermediate Re) along with the unit cell approach. A good agreement between experiments and CFD results was obtained in the range of superposition. Results showed that woven spacers guarantee a better mixing than overlapped ones, especially in the low to intermediate Re range, thus resulting in Nusselt numbers 2.5–3 times higher. On the other hand, the less disturbed flow field induced by overlapped spacers was found to yield friction coefficients up to 4 times lower, thus allowing lower pumping costs. The choice between the two configurations depends crucially on the relative importance attributed to savings in membrane surface area and in pumping energy for any specific application.