Multiple indicators have been used to assess the degree of exposure of fish to anthropogenic chemicals in their stream habitats. We hypothesized that brook stickleback (Culaea inconstans) in a headwater stream receiving urban and agricultural runoff (South Fork Whitewater River, SFWR) would exhibit poorer condition, reduced reproductive fitness, and a greater left side to right side morphological asymmetry (i.e., fluctuating asymmetry or FA) than fish from a nearby headwater stream with a forested drainage basin (Garvin Brook). Male and female fish were collected from both streams just prior to spawning in 2013–2015. In 2013 and 2014, fish were assessed for overall condition (Fulton’s K), internal measures of condition (hepatosomatic index, HSI) and reproductive fitness (gonadosomatic index [GSI], total oocyte count, and oocyte mass). In 2015, measurements of head length, jaw length, eye diameter, pectoral fin length, and pelvic fin length were made on both sides of each fish for assessing degree of FA. We observed declining condition with fish size, increased liver size, and reduced oocyte counts and oocyte size in female brook stickleback in SFWR relative to those from Garvin Brook. SFWR females had significantly higher FA than Garvin females for all structures assessed, except pelvic fin length. FA also was slightly higher for all structures in SFWR males compared to Garvin males, but differences were not significant. A composite FA index combining all measurements from an individual fish into a single value displayed highly significant differences for female fish (SFWR FA >> Garvin FA), but not for male fish (SFWR FA = Garvin FA). Exposure of brook stickleback to reduced water quality in SFWR during early development appears to increase morphological asymmetry in female (but not male) fish, and continuing exposure to compromised water quality throughout life impacts both general condition and reproductive fitness of stickleback, especially older female fish, in SFWR.