Aims: To investigate the anti-HSV and anti-inflammatory effects of a standardized ethyl acetate extract (SEAE) prepared with the stem bark of Strychnos pseudoquina, along with two isolated compounds: quercetin 3-O-methyl ether (3MQ) and strychnobiflavone (SBF). Methods and Results: The mechanisms of action were evaluated by different methodological strategies. SEAE and SBF affected the early stages of viral infection and reduced HSV-1 protein expression. Both flavonoids elicited a concentration-dependent inhibition of monocyte chemoattractant protein-1 (MCP-1), whereas 3MQ reduced the chemokine release more significantly than SBF. Conversely, both compounds stimulated the production of the cytokines TNF-alpha and IL-1-beta in LPS-stimulated cells, especially at the intermediate and the highest tested concentrations. Conclusions: SEAE and SBF interfered with various steps of HSV replication cycle, mainly adsorption, postadsorption and penetration, as well as with beta and gamma viral proteins expression; moreover, a direct inactivation of viral particles was observed. Besides, both flavonoids inhibited MCP-1 selectively, a feature that may be beneficial for the development of new anti-HSV agents. Significance and Impact of the Study: The results indicated that the samples present anti-HSV and anti-inflammatory activities, at different levels, which is an interesting feature since cold and genital sores are accompanied by an inflammation process.