Feijoa sellowiana Berg. fruit, widely used for human consumption, is well appreciated for its good nutritional characteristics and for its pleasant flavor and aroma. In a prior study we showed that the acetonic extract of F. sellowiana fruit exerts a potent antibacterial activity against some Gram-positive and Gram-negative bacterial strains. To confirm and deepen the study on antimicrobial activities of the fruit, in this article we analyze the same extract for its effectiveness against different kind of bacteria both as American Type Culture Collection standard strains and clinically isolates, the Gram-negative Helicobacter pylori, and three fungal strains (one human pathogen and two phytopathogens). Our aim was also to isolate and define the active component(s) involved in the antibacterial, antifungal, and anti-H. pylori activity of the extract through activity-guided fractionation procedures. This allowed us to identify flavone as the active compound of F. sellowiana fruit. Flavone showed a high antibacterial activity against the nine standard bacterial strains tested and the matched clinically isolated bacterial strains and was significantly more active against H. pylori than metronidazole. Pseudomonas aeruginosa, Proteus mirabilis, and Proteus vulgaris from both standard and clinical sources showed a very high sensitivity to flavone (minimal inhibitory concentration [MIC] = 1.95 microg/mL for standard strains and 3.9 microg/mL for clinical isolates of the three bacteria), showing also good minimal bactericidal concentration values. Among fungi tested, Rhyzoctonia solani was the most sensitive strain to the action of flavone (MIC = 62.5 microg/mL).