Vitamins are essential nutrients to the body needs in small amount to work properly. The attenuation coefficient is an important parameter for characterizing the penetration and diffusion of x-rays and gamma rays in biological material. The measurement of mass, linear and mean free path of biological samples viz. thiamine, adenine, pyridoxine and ascorbic acid in the energies 0.122, 0.356, 0.511, 0.662, 0.840, 1.170, 1.275, 1.330 MeV by using NaI(Tl) scintillation detector. The measured values of mass, linear attenuation coefficient and mean free path showed good agreement with theoretical values.