Although it is widely assumed that business activity is dependent on flows of ecosystem services (ES), little evidence is available with which to evaluate this contention. To address this knowledge gap, we conducted a questionnaire survey of business dependencies on twenty-six different ES in the English county of Dorset, where the environment supports a significant component of the local economy. Responses were received from 212 businesses across twenty-eight sectors. While virtually all businesses (98%) were familiar with the concept of ES, dependency on ES was highly divided with 50% of businesses surveyed claiming no dependence on any ES flows. The highest businesses dependencies reported in this study were for regulating services with the ES of water quality and waste water treatment being of particular importance to businesses. The results however, advised that greater efforts are needed in highlighting the indirect benefits provided by Dorset’s ecosystems, with eight business sectors (58% of respondents) claiming no or little dependence on supporting and habitat services including the ES of biodiversity, habitats for species and maintenance of genetic diversity. Many businesses also indicated little or no dependence on the globally important ES of pollination and soil condition, which may reflect a lack of awareness of dependencies occurring upstream of their value chains. At the sector level, businesses directly involved in protecting, extracting, or manufacturing raw materials were found to be more dependent on provisioning, regulatory and supporting ES than those operating in the service sector who favored cultural ES. These results highlight the value of assessing business dependencies on ES flows, which could usefully inform environmental management and accounting systems and improve monitoring of business performance, and thereby contribute to achievement of sustainability goals.