Background: Efficient iontophoretic transdermal delivery of hydrophilic drug molecules requires selection of appropriate aqueous formulation. In this study, oil/water cream and gel formulations were investigated for iontophoretic transdermal delivery of acyclovir (ACV), a model hydrophilic small-drug molecule, across hairless rat skin on Franz diffusion cells. Results: Iontophoresis (0.2 mA/cm2) enhanced ACV delivery from both 5% cream (pH 6.8) and 4% gel (pH 11) formulations. However, sixfold higher drug levels were delivered across the skin using gel formulation (12.25 ± 4.04 µg/cm2) as compared with cream formulation (2.03 ± 0.05 µg/cm2). Significantly higher drug levels were delivered when iontophoresis was performed at higher current density (0.32 mA/cm2; p < 0.05). Influence of formulation co-solvents (glycerin and propylene glycol) on drug delivery was also investigated in vitro using Franz cells and in vivo in hairless rats using microdialysis. Conclusion: Iontophoretic transdermal delivery of ACV was feasible and dependent on the selection of formulation components and delivery parameters.