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The increasing presence of aquatic contaminants, such as the pharmaceutical fluoxetine, has raised concerns over potentially disrupting effects on several aspects of fish reproduction. However, the effects of fluoxetine on reproductive and paternal behavior in fish remain understudied, particularly at environmentally relevant concentrations. In the current study, we therefore tested the hypothesis that waterborne fluoxetine at an environmentally relevant concentration (540 ng/l), disrupts specific reproductive and paternal behaviors in male Siamese fighting fish at distinct reproductive phases. A pre-post test design was adopted to investigate specific behavioral responses at the individual fish level in response to male conspecific intruders at two different distances from the nest across four distinct reproductive phases (before bubblenest construction, following bubblenest construction, after spawning and after hatching of the larvae). In the control specimens, the measured behaviours were not different between the spawning times and among the interactions in either distance to nest at the different reproduction phases. Our results indicate that fluoxetine specifically disrupts characteristic paternal territorial aggression behaviour only after spawning and hatching of the larvae, while male behaviour in previous reproductive phases is unaffected by fluoxetine exposure. Results of comparison between males at 1st spawning and specimens exposed to fluoxetine at 2nd spawning showed that the first reaction of the nest-holding males to the intruders, duration of fin spreading, number of bites, and 90° turn, and the frequency of sweeps were different between the spawning times after spawning or hatching of embryos. However, interaction of spawning time and reproduction phase was significant on biting behaviour. These results demonstrate that fluoxetine exposure at environmental concentrations negatively affects territorial defense behaviour in fighting fish during parental care after larval hatching, which may have possible implications on reproductive success and population dynamics.