Fluid dynamics has been shown to alter ecologically important behaviors of aquatic organisms orienting to distant chemical sources. Because the fluid dynamics and chemical plumes change across hydraulic environments, it is unclear which of these factors influence orientation behavior more. This study examined how alterations in chemical signal structure, through changes in source spatial arrangement, affect chemically mediated search behavior. Microelectrochemical measurements of tracer molecules revealed that source arrangment significantly alters the downstream fine-scale structure of chemical plumes. Flume hydrodynamic characterizations (as measured with laser Doppler velocimetry) also differed among source arrangements; however, differences were minor and existed only at select upstream regions of the flume. Crayfish (Orconectes virilis) found the source faster and spent less time in refuges when sources were separated, compared with sources together. Similar numbers of crayfish found the source regardless of source arrangement. Crayfish searched more efficiently with increased spatial complexity at the sources. These results supported the hypothesis that spatial and temporal dynamcis of chemicals within plumes contain important information that organisms use during olfactory-mediated orientation in streams.