Satureja montana essential oil is a natural substance able to inhibit the growth of several pathogens. This antimicrobial effect is often attributed to its ability to penetrate cellular structures and disrupt them. Although these properties are recognized as playing a key role in the mechanism of action of this substance, many unresolved issues still exist, and fundamental studies focused on such aspects are scarce. In this framework, we investigated the interaction of SEO with lipid monolayers, which represent simplified models of cell membranes, using the Langmuir monolayer technique, complemented by fluorescence anisotropy and differential scanning calorimetry on lipid bilayers. By focusing on packing conditions that approximate those of biological membranes and using lipids with different polar heads and structures, such as the ones occurring in bacterial membranes, we aim to clarify the effect of this essential oil on the lipid membrane. Our results show that Satureja montana essential oil consistently manages to insert into the membrane and interfere with the lipid–lipid interactions, thereby altering the lipid packing and significantly increasing the membrane fluidity, depending on the oil concentration and the nature of the lipid.