We hypothesized that host-associated microorganisms can alter host behavior by modifying perfluorooctane sulfonamides to produce perfluorooctanesulfonic acid (PFOS) or other potentially neuroactive metabolites. Zebrafish larvae (Danio rerio) were exposed to PFOS (0.28–5 μM), N-ethyl perfluorooctane sulfonamide (N-EtFOSA, 0.07–1.25 μM), or perfluorooctane sulfonamido ammonium iodide (PFOSAmS, 0.83–15 μM) from 5 to 6 days post fertilization (dpf). This resulted in altered dark-phase swimming behavior at 8 dpf. Exposure to PFOS, or N-EtFOSA, but not PFOSAmS caused a similar shift in community structure. We exposed microbiome-depleted (axenic, AX), conventionally colonized (CC), or axenic conventionalized on day 1 (AC1) zebrafish larvae to N-EtFOSA (0.22–0.7 μM) from 5 to 6 dpf. In comparison to CC and AC1 larvae, AX-larvae did not exhibit concentration-dependent hypoactivity at 8 dpf. Chemical analysis at 7 dpf revealed a significant increase in N-EtFOSA levels in AX-larvae and AX-water samples, relative to colonized cohorts. The main metabolite detected was perfluorooctane sulfonamide (FOSA), which was not microbiome-dependent. Perfluorooctane sulfonamidoacetic acid (FOSAA) was detected at lower levels in AX-larvae, relative to colonized groups. This study revealed that the presence of the microbiome enhanced N-EtFOSA-dependent behavior effects at the level of the host. This supports the concept that chemical–microbiome interactions can influence host phenotypic outcomes.