This study aimed to achieve an in vitro quantification of the effects of composition and formulation factors on the killing rates of alcohol-based hand sanitizers. The killing rates of 85% ethyl alcohol (ET) and isopropyl alcohol (IPA) were studied under different conditions such as pH, electrolyte concentration, or inclusion of herbal extracts (cucumber, carrot, and aloe vera), a quaternary ammonium compound, or thickener over different time intervals. Changes in the activities were retested after 3 months as an indication of stability. From two-way ANOVA, both the time of exposure and the sanitizer type affected the activity against Staphylococcus aureus (P = 0.001 for both alcohols), whereas for Escherichia coli, time of exposure was significant (P = 0.027), while sanitizer type was less significant (P = 0.063). Extreme pHs, the presence of ions, and the inclusion of additives such as benzalkonium chloride (BAC), plant extracts, or carbomer impacted the 3-month activity of the samples differently. Important differences existing in the activities of ET and IPA, as a function of formulation factors or use conditions have been quantified using in vitro methods. Formulations should best be tailored for particular purposes and the all-purpose hand sanitizer may not exist. Graphic Abstract