Silica mesoporous films with orthorhombic Fmmm mesostructure have been produced using evaporation induced self-assembling via dip-coating. A block copolymer, Pluronic F-127, has been used as the organic template and has been removed from the films by thermal calcination. The block copolymer templated films have been characterized as a function of the calcination temperature, in the range 150–550 °C, by grazing incidence small angle X-ray scattering (GI-SAXS) and Fourier transform infrared spectroscopy. Thermal dehydroxylation formed isolated and geminal silanols on the pore surface and the removal of the organic template has increased the water absorption within the pores. The calcination has induced a shrinkage of the pores in the direction normal to the substrate, as it has been observed by GI-SAXS. Humidity sensors based on the mesostructured silica films as sensing membrane have been fabricated using alumina substrates with interdigitated gold fingers. The electrical response of the sensors in different relative humidity (RH) conditions has been measured. In the considered RH range the sensor revealed a high sensitivity with more than 4 orders of magnitude change in the current. Little hysteresis loop has been observed in the electrical response recorded increasing and reducing the RH value. The films calcined between 350 and 450 °C showed the best performances in term of stability, hysteresis and reproducibility of the response. The current response has been correlated with the water absorption within the pores.