Costs and benefits of territorial defense may vary with the number of intruders that a resident simultaneously encounters. Our study examined whether or not aggressive or escape behavior of female Plethodon cinereus would increase as the number of intruders simultaneously entering her territory increased from zero to one to two. We allowed 30 focal female P. cinereus to establish territories in laboratory chambers and tested each female in five randomly sequenced treatments: (1) one female intruder; (2) two female intruders; (3) one male intruder (4) two male intruders; and (5) a surrogate control (no live intruders). We recorded the behavior of the focal salamanders in response to the intruders. Across the five treatments, females differed significantly in amount of time in threat and escape behaviors and in total number of bites. Post hoc comparisons found that relative to the control females displayed significantly more time in threat behavior toward one male intruder and significantly more time in escape behavior in the presence of two male intruders, but they exhibited no significant differences toward one or two female intrudes. The post hoc comparisons for bites found no significant differences. We infer that for female P. cinereus the benefit of guarding prey in a territory outweighed the cost of defense (possible injury) when a single male was introduced into her territory, but the cost of defense exceeded the benefit when two male intruders were simultaneously introduced into her territory.