Herpes simplex virus type 1 (HSV-1) causes keratoconjunctivitis and encephalitis, with nucleoside analogues such as acyclovir (ACV) serving as standard treatments. However, rising drug resistance underscores the need for agents with novel mechanisms. In this study, phytochemical investigation led to the obtainment of several phloroglucinol derivatives from Rhodomyrtus tomentosa. Subsequent phenotypic screening demonstrated compound 6 as the most promising HSV-1 inhibitor. To facilitate lead optimization, we synthesized 44 analogues of 6 to elucidate the structure-activity relationships, and 24 analogues exhibited comparable potency to 6 against both HSV-1 and ACV-resistant viruses. Preliminary mechanistic studies revealed that 6 can inhibit viral replication by blocking host cell factors involved in serine 5-phosphorylated RNA polymerase expression. Furthermore, 6 also showed efficacy in vivo against HSV-1 infection via topical administration. These findings highlight a new type of anti-HSV-1 inhibitors with a mechanism distinct from that of ACV, offering new avenues for anti-HSV-1 drug development.