Biofuels and conventional fuels differ in terms of their evaporation rates, permeation rates, and exhaust emissions, which can alter exposures of workers, especially those in the fuel refining and distribution industries. This study investigated the permeation of biofuels (bioethanol 85%, biodiesel 20%) and conventional petroleum fuels (gasoline and diesel) through gloves used in occupational settings (neoprene, nitrile, and Viton) and laboratories (latex, nitrile, and vinyl), as well as a standard reference material (neoprene sheet). Permeation rates and breakthrough times were measured using the American Society for Testing and Materials F739-99 protocol, and fuel and permeant compositions were measured by gas chromatography/mass spectrometry. In addition, we estimated exposures for three occupational scenarios and recommend chemical protective clothing suitable for use with motor fuels. Permeation rates and breakthrough times depended on the fuel-glove combination. Gasoline had the highest permeation rate among the four fuels. Bioethanol (85%) had breakthrough times that were two to three times longer than gasoline through neoprene, nitrile Sol-Vex, and the standard reference materials. Breakthrough times for biodiesel (20%) were slightly shorter than for diesel for the latex, vinyl, nitrile examination, and the standard neoprene materials. The composition of permeants differed from neat fuels, e.g., permeants were significantly enriched in the lighter aromatics including benzene. Viton was the best choice among the tested materials for the four fuels tested. Among the scenarios, fuel truck drivers had the highest uptake via inhalation based on the personal measurements available in the literature, and gasoline station attendants had highest uptake via dermal exposure if gloves were not worn. Appropriate selection and use of gloves can protect workers from dermal exposures; however, current recommendations from the National Institute for Occupational Safety and Health should be revised to account for contemporary fuel formulations that routinely contain ethanol.