The present study was conducted to assess the toxicity of the water-accommodated fraction (WAF) and the chemically enhanced WAF (CE-WAF) of selected crude oils for both weathered and fresh oil. Test organisms included two standard test species, Mysidopsis bahia and Menidia beryllina, and a commercially important Florida marine fish, Sciaenops ocellatus. Tests ascertaining LC50 values were conducted under continuous exposure and spiked (declining exposure using flow-through toxicity chambers) conditions using Venezuelan Crude Oil (VCO), Prudhoe Bay Crude Oil (PBCO), and COREXIT® 9500 dispersant on the above species. Data suggest that the dispersant is less toxic than the WAF and CE-WAF of the tested crude oils. The toxicity of the CE-WAF of fresh VCO is similar to that of other oils under continuous exposure conditions, but may be slightly more toxic to some species under spiked exposure conditions. The CE-WAF of fresh VCO appears to be less toxic than the corresponding WAF for M. bahia, M. beryllina, and S. ocellatus. Fresh VCO appears to be much more toxic to M. bahia and M. beryllina than weathered VCO in spiked exposure tests for both the WAF and CE-WAF. The WAF of PBCO is apparently less toxic to the test organisms than the corresponding WAF of fresh VCO. The LC50 values of M. bahia with CE-WAF fractions of both fresh VCO and PBCO are similar, while the same PBCO CE-WAF fraction is less toxic for M. beryllina than fresh VCO CE-WAF. The toxicity of oils and dispersants were lowest in the spiked exposure weathered oil tests, which may be most representative of an oil spill under natural environmental conditions.