In this study, we investigated, in vitro, the effects of petroleum hydrocarbons on the phospholipid ester-linked fatty acid composition of Corynebacterium sp. Strain 8. The usual ratio of monounsaturated fatty acids E/Z (or trans/cis) was calculated. This ratio led to unexpected results because we found similar values for growths on either a hydrophobic substrate (crude oil) or a soluble carbon source (rich medium). The use of such an indicator seemed limited for monitoring an environmental stress, so we proposed an index based on the homeoviscous adaptation theory. A membrane viscosity index was defined and applied to Corynebacterium sp. Strain 8 (in vitro growth) and to a sedimentary community (in situ experiment). The results allowed us to estimate the membrane fluidity of both an isolated strain and a bacterial community in accordance with the medium hydrophobicity.