A set of parallel microfluidic channels behaving as a diffraction grating operating in the Raman-Nath regime has been fabricated and studied. The diffraction efficiency of such structure can be tuned by selecting a liquid with a particular refractive index and/or optical anisotropy. Alternatively the optical properties of the liquid can be characterised by measuring the diffraction efficiency and the state of polarization of the diffracted beam. In this work, the microfluidic channels under study have been filled with penicillin molecules dissolved in water. Due to the chirality of the penicillin, the liquid has been found to have circular birefringence of 2.14 × 10−7. The addition of the anisotropic liquid modifies the polarization properties of the microfluidic diffraction grating. The diffraction efficiency of the grating has been characterised for different probe beam wavelengths and states of polarization. Currently the diffraction efficiency of the device is low – 1.7%, but different approaches for its improvement have been discussed.