The spatial variability in the food chain structure of an estuary environment (Nanakita estuary, Japan) was investigated using fatty acid. Potential organic matter sources (terrestiral plants, macroalgae, benthic microalgae, dinflagellates and bacteria), sedimentary organic matters and benthic invertebrates (Nuttallia olivacea and Nereidae) were sampled in four locations with different tidal flat type. The main objective of the present study was to determine the origin of sediment and the food sources of N. olivacea and Nereidae along with small-scale spatial variability. The origin of sedimentary organic matters were mainly the fatty acid of bacteria and benthic microalgae. Especially, The organic matter of terrestrial plant origin was found the highest in station C. The diets of N. olivacea and Nereidae were found to be dominated by diatoms and terrestrial plants. Whereas, macroalgae and dinoflagellates showed little influence to benthic invertebrates. Moreover, according to principal component analysis, it is showed that benthic invertebrates in the same region are using the same food without relation with species. On the other hand, the N. olivacea and Nereidae of station D clearly contrasts with station B in terms of main food sources. From these results, it is suggested that food competition of benthic invertebrates revealed high a connection between small-scale spatial variability and food source in estuary.