This article deals with the validation of and first measurements with a newly constructed flowing afterglow selected ion flow tube (FA-SIFT) instrument. All reactions were studied in He buffer gas at a pressure of 1.43 hPa and a temperature of 298 K. The validation consisted of the study of the gas-phase ion/molecule reactions of methanol and ethanol (M) with the reactant ions H 3O +·(H 2O) n ( n = 0–3), MH +, M 2H +, and MH +·H 2O and the reactions of MH + with H 2O. Obtained results are compared with available literature data and with calculated collision rate constants. The validated FA-SIFT has subsequently been used to characterize the reactions of the unsaturated biogenic alcohols 2-methyl-3-buten-2-ol, 1-penten-3-ol, cis-3-hexen-1-ol and trans-2-hexen-1-ol (ROH) with H 3O +·(H 2O) n ( n = 0–3) as well as the secondary reactions of the H 3O +/ROH product ions with H 2O (hydration) and ROH in view of their accurate quantification in ambient air samples with medium-pressure chemical ionization mass spectrometry (CIMS) instrumentation using H 3O + reactant ions. Whereas water elimination following proton transfer was found to be the main mechanism for all H 3O +/ROH reactions studied and for the H 3O +·H 2O/ trans-2-hexen-1-ol reaction, all other H 3O +·(H 2O) n /ROH ( n = 1, 2) reactions proceeded by multiple reaction mechanisms. H 3O +·(H 2O) 3 reactions proceeded mainly (C 6 alcohols) or exclusively (C 5 alcohols) by ligand switching followed by water elimination. Hydration of the H 3O +/ROH product ions was observed whenever they contained oxygen. The secondary reactions with ROH were also found to proceed by multiple reaction pathways.