Household products often contain an antimicrobial agent such as biocides, polyhexamethylene guanidine (PHMG), triclosan (TCS), and propylene glycol (PG) as an excipient to dissolve the active ingredients. The skin sensitization (SS) potentials of each of these substances or mixtures of PHMG or TCS with PG have not been investigated through in vitro alternative test methods. The in vitro alternative assay called human Cell Line Activation Test (h-CLAT) served to address these issues. The h-CLAT assay was conducted in accordance with OECD TG 442E. On three independent runs, all the three substances were predicted to be sensitizers according to the SS positivity with relative fluorescence intensity of CD86 ≥ 150% and/or CD54 ≥ 200% at any tested concentrations. Mixtures of PHMG or TCS with PG at ratios of 9:1, 4:1, or 1:4 weight/volume were all positive in terms of SS potential. Since humans can be occupationally or environmentally exposed to mixtures of excipients with active ingredients of biocides, the present study may give insights into further investigations of the SS potentials of various chemical mixtures.