Ambrosia beetles (Coleoptera; Curculionidae; Scolytinae and Platypodinae) can cause severe damage to trees growing in plant nurseries, orchards and natural forests. Ethanol is emitted by stressed trees and represents an important cue used by ambrosia beetles to locate suitable hosts to infest. Ethanol also favors the growth of ambrosia beetles’ nutritional fungal symbionts and suppresses the growth of antagonistic fungi. An optimal concentration of ethanol in host tissues might maximize fungal growth and offspring production, but it is unclear if this optimal concentration varies among ambrosia beetle species. To investigate this mechanism, we injected five different concentrations of aqueous ethanol solution (5%, 25%, 50%, 75% and 90%) into the stems of container-grown oak trees, Quercus robur L. Modified Falcon tube chambers were used to confine four species of field-collected ambrosia beetles to the injected stems, namely, Anisandrus dispar, Xyleborinus saxesenii, Xylosandrus germanus, and Xylosandrus crassiusculus. Incidence of boring, ejected sawdust, gallery development, and offspring production were then quantified. The incidence of boring generally increased with increasing ethanol concentration for all four Scolytinae species tested. Ejected sawdust and offspring production increased with increasing ethanol concentration up to 90% for A. dispar and X. saxesenii; by contrast, an increasing trend up to 75% ethanol followed by a decrease at 90% ethanol was associated with X. germanus and X. crassiusculus. Our study highlights the key role of ethanol for ambrosia beetles, and showed that the optimal concentration maximizing colonization and offspring production can vary among species.