Diamond films were prepared on a pure Ti substrate using the microwave plasma assisted chemical vapor deposition (MW-PACVD) method. The effects of applying two types of gas mixtures (H2/CH4 and Ar/H2/CH4) during diamond deposition on the microstructure of a Ti substrate were studied. With H2/CH4 (196:4) gas mixture, during diamond film deposition, hydrogen diffused into the Ti substrate and led to significant microstructure coarsening and a severe loss in Charpy impact energy. Post dehydrogenation annealing at a temperature of 800°C, could not change the coarse structure of the substrate, thus there was no improvement in Charpy impact energy. With the application of Ar/H2/CH4 (180:16:4) gas mixture, a smooth and nano-crystalline diamond film was deposited, and there was a minimum change in the substrate microstructure and Charpy impact energy after diamond deposition. The above results provide useful information for the successful application of diamond films (on a Ti substrate) for biomedical and aerospace application.