[2] The harlequin darter displays a green coloration on its sides, with six or seven brown saddles present along the top of the body.

The name harlequin darter refers to mask-like pigmentation on the face, consisting of a suborbital bar and dark blotching on the head, breast, and body.

[6] The diet of the harlequin darter consists of benthic invertebrates, including larvae from midges, caddisflies, mayflies, and blackflies.

[11] Within its range, the harlequin darter prefers moderate- to fast-flowing riffles with fairly high water quality conditions;[10] but what sets it apart is its dependence on detritus and downed woody debris.

It is one of the few darter species reliably found within the main channel of the Mississippi River, and it often avoids smaller streams.

[4] Agricultural runoff tends to accumulate heavily into these large water bodies, greatly increasing nitrate and phosphate levels, creating an unfavorable environment for these darters.

[6] Also, companies along these larger bodies of water tend to remove vegetation and woody debris in riparian zones, further negatively affecting the harlequin darter.

Late spring through fall are generally spent in smaller streams, and during the colder months, movement occurs into larger reservoirs and other large bodies of water.

The Missouri Department of Conservation, one of the few that have developed best management practices for the harlequin darter, restricts the dates when work can be done near wetlands to protect harlequin darter breeding, leaves vegetation and woody debris in water bodies, puts up sediment controls such as silt fences, avoids the use of permanent dams that restrict movement, and avoids stream crossings by using culverts or detouring routes that cross streams where the harlequin darter resides.

[12] A biological status review conducted in Florida determined many of the same management problems, including woody debris removal, damming water bodies, turbidity, and sediment loads lead to the decrease in harlequin darter numbers.

[3] The harlequin darter exhibits some degree of seasonal movement, going from large rivers into smaller tributaries for part of the year,[12] so the removal of dams and other dispersal barriers would benefit this species.