In eusocial insects like Bald-faced hornets, Dolichovespula maculata, nest defense is essential because nests contain a large number of protein-rich larvae and pupae, and thus are attractive to nest predators. Our objectives were to investigate whether D. maculata exhibit pheromone-mediated nest defense, and to identify and field test any pheromone components. We tested for pheromone-mediated nest defense behavior of D. maculata by placing a paired box-apparatus near the entrance of D. maculata nests, and treating both boxes with a solvent control, or one of the two boxes with a solvent control and the other with either venom sac extract, the putative source of nest defense pheromone, or synthetic pheromone. The sound impulses caused by nest mates attempting to sting or strike the boxes were recorded for 3 min. Compared to the double-control treatment, the number of strikes increased 27-fold when one of the two boxes was treated with venom sac extract, providing evidence for an alarm response. The box treated with venom sac extract also induced a significantly greater proportion of strikes than the corresponding control box, providing evidence for a target-oriented response. Analyzing venom sac extract by gas chromatographic-electroantennographic detection (GC-EAD) and GC-mass spectrometry resulted in the identification of seven candidate pheromone components: (a) dimethylaminoethanol, (b) dimethylamino ethyl acetate, (c) 2,5-dimethylpyrazine, (d) N-3-methylbutylacetamide, (e) 2-heptadecanone, (f) (Z)-8-heptadecen-2-one, and (g) (Z)-10-nonadecen-2-one. Testing in paired-box bioassays blends of the nitrogen-containing volatile components a-d, the less volatile ketones e-g, or both (a-g), indicated that a-d primarily have an alarm function. The ketones e-g, in contrast, induced target-oriented responses, possibly marking the box, or potential nest predators, for guided and concerted attacks, or enhancing the alarm-inducing effect of the volatile pheromone components, as shown in honey bees. Comparing the behavioral effects of venom sac extract, blends a-d, e-g, and a-g, venom sac extract was most effective in triggering the full complement of alarm and target-oriented responses. These comparisons further suggested that a component is missing in the group of components that triggers the alarm rather than the target-oriented response.