The symmetry breaking effects for quantum tunneling of the magnetization in Mn12-acetate, a molecular nanomagnet, represent an open problem. We present structural evidence that the disorder of the acetic acid of crystallization induces sizable distortion of the Mn(III) sites, giving rise to six different isomers. Four isomers have symmetry lower than tetragonal and a nonzero second-order transverse magnetic anisotropy, which has been evaluated using a ligand field approach. The result of the calculation leads to an improved simulation of electron paramagnetic resonance spectra and justifies the tunnel splitting distribution derived from the field sweep rate dependence of the hysteresis loops.