In the most recent decades the introduction of unconventional machining processes allowed the development of micromachining techniques. In this work, the influence of material microstructures on the micromilling process was investigated. Ti6Al4V alloy was selected as workpiece material since it is a very common material for micro applications and because its duplex microstructure can be easily changed by proper thermal treatments. Four different microstructures (namely bimodal, fully equiaxed, fully lamellar and mill annealed) were obtained through recrystallization annealing treatments carried out at different times and temperatures. The mechanical properties of the samples were assessed by microhardness measurements. Nano-indentations were also performed on single grains to understand how the different hardness of phases and structures present in the Ti6Al4V alloy can affect the micromilling process. Microchannels using two flute flat end mills with a diameter equal to 200 µm were realized on the treated samples. Two different feed-per-tooth values were used during the tests. Cutting force, channel shape and burr dimension were investigated. Morphological and energy dispersive spectroscopy (EDS) analyses were performed on tools by means of a scanning electron microscope (SEM): in this way the phenomena mainly influencing the tool status were also identified. Lower cutting forces and reduced tool wear were observed when working fully lamellar microstructures compared to the other ones.