We have applied density functional theory and high-resolution transmission electron microscopy to investigate the relationship between chemically induced strain and charge transfer on the structural, electronic, vibrational, and thermoelectric properties of misfit layered cobaltites (M2CoO3)0.6CoO2 (M = Mg, Ca, Sr, Ba). The electron and phonon density of states are analyzed and rationalized by accounting for the effects of internal strain and charge transfer, and lay the foundations to disentangle these effects on a promising thermoelectric oxide material. We found that the choice of different interlayer cations has little effect on the magnetic properties, but it generates internal strain between the rock-salt M2CoO3 and hexagonal CoO2 subsystems, changing the hybridization of the cations with the environment. Increasing the mass of the cation leads to decoupling of the vibrations between the rock-salt and CoO2 subsystems so that heavier cations are predicted to enhance phonon scattering. On the other hand, applying compressive strain to the system, which corresponds to doping with smaller interlayer cations, is shown to enhance the Seebeck coefficient. The calculations suggest that thermoelectric efficiency of misfit layered cobaltites may be tuned by codoping the rock-salt layer with isovalent alkali earth cations.