Background Photodynamic therapy (PDT) delays ultraviolet (UV) radiation-induced squamous cell carcinomas (SCCs) in hairless mice. Efficacy may be enhanced by combining PDT with antineoplastic or pro-differentiating agents. We investigated if pretreatment with 5-fluorouracil (5FU), imiquimod (IMIQ) or calcipotriol (CAL) before PDT further delays tumor onset. Methods Hairless mice (n = 224) were exposed 3 times weekly to 3 standard erythema doses (SED) of UV radiation. Methyl-aminolevulinate (MAL)-PDT sessions were given on days 45 and 90 before SCC development. Three applications of topical 5FU, IMIQ or CAL were given before each PDT session. Fluorescence photography quantified protoporphyrin IX (PpIX) formation. Results PDT delayed UV-induced SCC development by 59 days (212 days UV-MAL-PDT vs. 153 days UV-control, P < 0.001). Pretreatment with 5FU, IMIQ or CAL before PDT did not further delay SCC onset compared to PDT alone (207 days UV-5FU-MAL-PDT, 215 days UV-IMIQ-MAL-PDT, 206 days UV-CAL-MAL-PDT vs. 212 days UV-MAL-PDT, P = ns). PpIX fluorescence intensified by 5FU-pretreatment (median 21,392 au UV-5FU-MAL-PDT, P = 0.011), decreased after IMIQ-pretreatment (12,452 au UV-IMIQ-MAL-PDT, P < 0.001), and was unaffected by CAL-pretreatment (19,567 au UV-CAL-MAL-PDT, P = ns) compared to MAL alone (18,083 au UV-MAL-PDT). Conclusions Short-term three-day pretreatment with 5FU, IMIQ and CAL before PDT does not further delay tumor onset in UV-exposed hairless mice.