The stocking of western mosquitofish Gambusia affinis for mosquito control negatively impacts native fishes with similar ecological requirements. In this study, a series of laboratory microcosm experiments was used to examine intra and interspecific agonistic behavioral interactions (e.g., chases and nips) between western mosquitofish and northern starhead topminnow Fundulus dispar, northern studfish F. catenatus, blackstripe topminnow F. notatus, and banded killifish F. diaphanous at three fish densities and in the presence/absence of vegetation. Western mosquitofish exhibited more agonistic behaviors than the four topminnow species and caused a change in topminnow behavior in mixed-species microcosms. Mosquitofish were aggressive toward conspecifics, with most of the chases and nips occurring at the highest densities and when vegetation was absent. Topminnows exhibited few agonistic behaviors toward conspecifics, but intraspecific chasing and nipping did occur when exposed to mosquitofish. Agonistic behaviors by topminnows toward mosquitofish occurred infrequently, and mosquitofish initiated almost all of the chases and nips. While all four topminnow species were attacked by mosquitofish, northern starhead topminnow and banded killifish were chased and nipped more frequently than the other topminnow species. These two topminnows exhibited the most behavioral changes and fin damage and one northern starhead topminnow died following mosquitofish attacks. Based on these results, it appears that the stocking of western mosquitofish into primary and connecting waterways could have negative impacts on native topminnow species that occur in these systems.