Dispersants are a preapproved chemical response agent for oil spills off portions of the U.S. coastline, including the Texas—Louisiana coast. However, questions persist regarding potential environmental risks of dispersant applications in nearshore regions (within three nautical miles of the shoreline) that support dense populations of marine organisms and are prone to spills resulting from human activities. To address these questions, a study was conducted to evaluate the relative toxicity of test media prepared with dispersant, weathered crude oil, and weathered crude oil plus dispersant. Two fish species, Cyprinodon variegatus and Menidia beryllina, and one shrimp species, Americamysis bahia (formerly Mysidopsis bahia), were used to evaluate the relative toxicity of the different media under declining and continuous exposure regimes. Microbial toxicity was evaluated using the luminescent bacteria Vibrio fisheri. The data suggested that oil media prepared with a chemical dispersant was equal to or less toxic than the oil-only test medium. Data also indicated that continuous exposures to the test media were generally more toxic than declining exposures. The toxicity of unweathered crude oil with and without dispersant was also evaluated using Menidia beryllina under declining exposure conditions. Unweathered oil-only media were dominated by soluble hydrocarbon fractions and found to be more toxic than weathered oil-only media in which colloidal oil fractions dominated. Total concentrations of petroleum hydrocarbons in oil-plus-dispersant media prepared with weathered and unweathered crude oil were both dominated by colloidal oil and showed no significant difference in toxicity. Analysis of the toxicity data suggests that the observed toxicity was a function of the soluble crude oil components and not the colloidal oil.