Microcirculatory blood flow can be measured using a laser Doppler flowmetry (LDF) probe. However, the readings are affected by the tissue’s optical properties (absorption and scattering coefficients, µa and µs ) and probe geometry. In this study the influence of optical properties [µa?(0.053,0.23) mm-1,µs?(14.7,45.7) mm-1] on LDF perfusion and LDF sampling depth was evaluated for different fiber separations. In vitro measurements were made on a sophisticated tissue phantom with known optical properties that mimicked blood flow at different depths. Monte Carlo simulations were carried out to extend the geometry of the tissue phantom. A good correlation between measured and simulated data was found. The simulations showed that, for fixed flow at a discrete depth, the influence of µs or µa on LDF perfusion increased with an increase in flow depth and decreased with an increase in fiber separation. For a homogeneous flow distribution, however, the perfusion varied 40% due to variations in the optical properties, almost independent of the fiber separation (0.23–1.61 mm). Therefore, the effect in real tissue is likely to vary due to the unknown heterogeneous blood flow distribution. Further, the LDF sampling depth increased with a decrease in µs or µa and an increase in fiber separation. For fiber separation of 0.46 mm, the e-1 sampling depth ranged from 0.21 to 0.39 mm.