Benthic diatoms are common phototrophic organisms of lotic and lentic environments and have the potential to be used as in-situ indicator communities of herbicide pollution. Since many herbicides mode of action is light dependent, such as the photosystem II inhibitors (PSII), light conditions during exposure may alter which species are most at risk of herbicide toxicity in the field. There have been no studies investigating light effects on the relative herbicide sensitivity of freshwater benthic diatoms. Additionally the potential for herbicides of differing modes of action to alter the relative sensitivity of benthic diatoms within a natural benthic community is not well understood. The results of two experiments are presented and discussed, the first investigates whether relative sensitivity is altered by herbicide mode of action, and the second compares the relative sensitivities of multiple benthic diatom taxa under different light exposure scenarios exposed to either Atrazine or Glyphosate. Natural benthic diatom communities were collected in-situ and then exposed to herbicides in rapid toxicity tests. There were significant interactions between light level and herbicide concentration response in 4 out of 31 taxa; Ulnaria sp., Gomphonema cf. olivaceum, Eunotia sp., and Navicula cf. veneta. However, in no cases did sensitive species appear to be tolerant, or vice versa, under altered light conditions. The relative herbicide sensitivity of benthic diatoms was independent of herbicide mode of action. This indicates freshwater benthic diatoms may be a suitable indicator for detecting toxicity of multiple herbicides, including those with differing modes of action.