<p>Antiviral-resistant herpes simplex virus (HSV) infections predominantly occur in immunocompromised individuals and represent a major therapeutic challenge. We conducted a retrospective observational study to describe the clinical characteristics and genotypic resistance profiles of patients with antiviral-resistant HSV infections managed at our center between January 2016 and December 2023. Genotypic resistance testing was performed by sequencing the <i>UL23</i> (thymidine kinase) and <i>UL30</i> (DNA polymerase) genes at the French National Reference Centre for Herpesviruses (Paris, France). Ten adult patients with confirmed resistance to at least one antiviral agent were included. Seven patients were infected with resistant HSV-1 strains, including five solid organ transplant (SOT) recipients, one patient with pemphigus vulgaris receiving immunosuppressive therapy, and one immunocompetent individual. Three people living with HIV (PLHIV) had resistant anogenital HSV-2 infections. HSV-1 infections presented with diverse clinical manifestations, including oral lesions (<i>n</i> = 4), pulmonary involvement (<i>n</i> = 3), keratitis (<i>n</i> = 1), cutaneous lesions (<i>n</i> = 1), and esophagitis (<i>n</i> = 1), with three SOT recipients exhibiting multisite disease. Clinical samples were obtained from corneal, oral, bronchoalveolar lavage, genital, and esophageal sites. Nine patients harbored <i>UL23</i> mutations associated with acyclovir resistance, including one previously undescribed mutation (I54K) confirmed by phenotypic assays. These mutations comprised amino acid substitutions (<i>n</i> = 3), frameshift mutations (<i>n</i> = 1), and premature stop codons (<i>n</i> = 5). One patient presented a <i>UL30</i> mutation (R628C) conferring resistance to both acyclovir and foscarnet. All patients had prior or ongoing antiviral exposure, mainly to acyclovir. The mean time to resistance detection in SOT recipients was 172 days post-transplantation, including three cases occurring despite antiviral prophylaxis. Genotypic resistance correlated with clinical failure of empirical acyclovir in eight cases. Consequently, four SOT recipients received second-line treatment with foscarnet, with clinical improvement of lesions observed in three cases, while treatment was discontinued in one due to nephrotoxicity. Among PLHIV, infections included recurrent genital herpes and atypical pseudo-tumoral lesions, managed with foscarnet or adjunctive topical cidofovir or imiquimod. These findings highlighted the importance of genotypic resistance testing to guide management in cases of suspected antiviral failure.</p>